3-oxa-8-azabicyclo[3.2.1]octane derivatives and their use in the treatment of cancer and hemoglobinopathies

ABSTRACT

wherein: n is 1 or 2; RN is H or Me; R1 is optionally one or more halo or methyl groups; R2a and R2b are independently selected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH2OH; R2c and R2d (if present) are independently selected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH2OH; R3a and R3b are independently selected from H and Me; R4a is selected from OH, —NH2, —C(═O)NH2, and —CH2OH; R4b is either H or Me; R5 is either H or Me; m is 1 or 2; q is 0 or 1; R11a, R11b, R11c and R11d are independently selected from H, halo, C1-4 alkyl, C1-4 fluoroalkyl, C3-4 cycloalkyl, C1-4 alkyloxy, NH—C1-4 alkyl and cyano; R12a and R12b are independently selected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH2OH; R12b and R12d are independently selected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH2OH; R12e is H or Me; R13a and R13b are independently selected from H and Me; R14 is either H or Me; R16a and R16b are independently selected from H and Me; R6 is selected from H, OMe, and OEt.

The present invention relates to amino-bicyclic compounds and their useas pharmaceuticals, and in particular, in treating cancer andhemoglobinopathies.

BACKGROUND TO THE INVENTION

Post-translational modification of proteins is a hallmark of signaltransduction where cells are able to react quickly to changes or eventsin the cellular environment. Post-translational modification of proteinsexpands the structural and functional diversity of the proteome. Therole of acetylation and phosphorylation of proteins has been extensivelystudied as highly reversible reactions for fine-tuning responses toexternal stimuli or changes in the environmental conditions. Recently,the importance of other types of protein modifications, includingubiquitination and methylation has begun to be recognized.

The methylation of proteins and the enzymes that carry out thesereactions has increased the dimensions of gene regulation by markinggenes that are transcriptionally active or silenced. Protein methylationcan occur on amino acids such as lysine, arginine, histidine, orproline, and on carboxy groups.

Arginine methylation of mainly nuclear proteins is an importantpost-translational modification process involved in structuralremodelling of chromatin, signal transduction, cellular proliferation,nucleocytoplasmic shuttling, translation, gene transcription, DNArepair, RNA processing, or mRNA splicing.

Methylation of proteins at arginine residues is catalysed by ProteinArginine Methyltransferase enzymes. The Protein Arginine MethylTransferase (PRMT) family of enzymes are evolutionarily conservedbetween organisms but differ in the number of members in differentorganisms.

There are eleven members of the human PRMT family, eight of which haveknown enzymatic activity and target substrates. With the exception ofPRMT2 and two recently identified putative PRMT genes (PRMT10 andPRMT11), all remaining proteins of the family possess enzymatic argininemethylation activity.

PRMTs are subdivided into two types based on the methylation that theycatalyse at the guanidinium group of arginine residues of substrateproteins. There are three nitrogens in the guanidinium group,potentially all of which could be methylated; the two ψ-guanidinonitrogen atoms and the internal δ-guanidino nitrogen atom.Mono-methylation and dimethylation of arginine (MMA and DMA) is found inmammalian cells at one or both of the two ψ-guanidino nitrogen atoms;dimethylation may be either symmetric or asymmetric. The thirdmethylated arginine is generated by monomethylation of the internalδ-guanidino nitrogen atom of arginine and has so far been documentedonly in yeast proteins. Type I PRMT enzymes catalyse the formation ofMMA and asymmetric dimethylarginine by di-methylating the same nitrogenatom of the guanidinium group, whereas Type II PRMT enzymes catalyse theformation of MMA and symmetric di-methylarginine by mono-methylatingeach of the terminal nitrogen atoms. Type III enzymes methylate theinternal δ-guanidino nitrogen atom. Of the eight well characterisedhuman PRMTs, PRMT1, 3, 4, 6 and 8 are Type I enzymes, and PRMT5, 7 and 9are Type II enzymes.

PRMTs catalyse the methylation of the guanidino nitrogen atoms ofarginine residues through the transfer of a methyl group from S-adenosylmethionine (SAM). A by-product of the enzymatic methylation step isS-adenosyl-L-homocysteine (AdoHcy), which is hydrolyzed to adenosine andhomocysteine by AdoHcy hydrolase (Krause et al., 2007).

PRMT5

PRMT5 (aka JBP1, SKB1, IBP72, SKB1his and HRMTIL5) is a Type II argininemethyltransferase, and was first identified in a two-hybrid search forproteins interacting with the Janus tyrosine kinase (Jak2) (Pollack etal., 1999).

PRMT5 plays a significant role in control and modulation of genetranscription. Inter alia, PRMT5 is known to methylate histone H3 atArg-8 (a site distinct from that methylated by PRMT4) and histone H4 atArg-3 (the same site methylated by PRMT1) as part of a complex withhuman SWI/SNF chromatin remodelling components BRG1 and BRM.

In addition to direct repressive histone marks induced by PRMT5, theenzyme's role in gene silencing is also mediated through the formationof multiprotein repressor complexes that include NuRD components, HDACs,MDB proteins and DNA methyltransferases, (Rank et al., 2010; LeGuezennec et al., 2006; Pal et al., 2003).

PRMT5 is involved in the methylation and functional modulation of thetumour suppressor protein p53. See (Berger, 2008; Durant et al., 2009;Jansson et al., 2008; Scoumanne et al., 2009). Most of the physiologicalfunctions of p53 are attributable to its role as a transcriptionalactivator, responding to agents that damage DNA. p53 status is wild typein approximately half of human cancer cases. These include 94% incervix, 87% in blood malignancies, 85% in bones and endocrine glands,and 75% of primary breast cancer. Restoration of p53 in cancer cellsharbouring wild type p53, by way of inhibiting mechanisms that suppressits function leads to growth arrest and apoptosis, and is regarded as apotentially effective means of tumour suppression.

p53 target genes have two alternative downstream effects: either theypause the cell cycle, allowing the DNA to be repaired, or, if repair isnot possible, they activate processes leading to apoptosis (programmedcell death). How p53 ‘chooses’ between these distinct outcomes is acentral question in the field of tumour biology.

p53 is replete with post-translational modifications. Phosphorylationwas one of the first post-translational modifications to be clearlydefined on p53. In the last decade it has become additionally clear thatp53 is modified not only by phosphorylation, but that it is extensivelymodified by lysine acetylation and methylation, among othermodifications. Indeed, besides histone proteins p53 is the most commonprotein substrate known for these post-translational modifications.However, despite the plethora of post-translational modifications, p53has not been identified, until recently, as a substrate for argininemethylation.

Jansson et al (Jansson et al., 2008) discovered that PRMT5 is physicallyassociated with a p53 cofactor called Strap. A co-factor complex thatcontains Strap et al binds to p53 in response to DNA damage. Jansson etal demonstrated that PRMT5 methylates p53 in vitro, and mapped the sitesof methylation (R333, R335 and R337). They developed an antibody thatspecifically detects p53 methylated on these sites and confirmed thatp53 is methylated in vivo. Jansson et al went on to show that p53methylation requires PRMT5 and is increased in response to etoposide, aDNA damaging agent.

The role of PRMT5 and p53 arginine methylation on cell cycle regulationand DNA damage response have been explored by both Jansson et al andScoumanne et al (Jansson et al., 2008; Scoumanne et al., 2009). Althoughsome differences are evident between the results from the two groups inrespect of cell cycle regulation in unperturbed cells (which may beascribed to cell type specific effects and/or the actual nature of theexperimental arrangements), both groups report similar results withrespect to the DNA damage response.

In response to DNA damage, caused by a variety of agents, includingdoxorubicin, camptothecin and UV light, and also in response totreatment with Nutlin-3, knockdown of PRMT5 results in an increase insub-G1 population and concomitant reduction in G1 cells and, in thepresence of p53, a significant increase in apoptosis. Knockdown of PRMT5also resulted in a reduced level of p21, a key p53 target gene thatregulates cell cycle arrest during the p53 response and MDM2, a p53 E3ubiquitin ligase, but not PUMA, NOXA, AIP1 & APAF1, p53 target geneslinked to apoptosis.

Knockdown of PRMT5 (but not PRMT1 or CARM1/PRMT4) results in decreasedp53 stabilisation, decreased basal p53 levels, and decreased p53oligomerisation, and also decreased expression of eIF4E a majorcomponent of translational machinery involved in ribosome binding tomRNA. Indeed, eIF4E is a potent oncogene, which has been shown topromote malignant transformation in vitro and human cancer formation.

Knockdown of PRMT5 would be expected to lead to a reduction in the levelof arginine methylated p53. Consistent with arginine methylation statusof p53 influencing the p53 response (reduced arginine methylationbiasing the response to proapoptotic), Jannson et al showed that a p53mutant in which each of the three critical arginine residues weresubstituted with lysine (p53KKK) retained the ability to induceapoptosis but its cell cycle arrest activity was significantlycompromised.

Moreover, pS3KKK also has a significantly reduced ability to inducetranscription of p21, by contrast with APAF1. The promoter bindingspecificity of wild-type p53 to key target genes is also significantlyaffected by arginine methylating status: Knockdown of PRMT5 results indecreased p53 binding to the promoter regions of the p21 and(intriguingly) PUMA genes, but does not affect p53 binding to thepromoter regions of NOXA or APAF1.

Taken together, it would seem that PRMT5 is a pro-survival factor, whichregulates cell proliferation in unstressed conditions and modulates thep53 response during DNA damage. In particular, knockdown of PRMT5,leading to a reduction in the levels of arginine methylated p53, appearsto bias the p53 DNA damage response to proapoptotic as opposed to cellcycle arrest.

PRMT5 is further linked to cancers in that it is aberrantly expressed inaround half of human cancer cases. PRMT5 overexpression has beenobserved in patient tissue samples and cell lines of Prostate cancer (Guet al., 2012), Lung cancer (Zhongping et al., 2012), Melanoma cancer(Nicholas et al., 2012), Breast cancer (Powers et al., 2011), Colorectalcancer (Cho et al., 2012), Gastric cancer (Kim et al., 2005), Esophagusand Lung carcinoma (Aggarwal et al., 2010) and B-Cell lymphomas andleukemia (Wang, 2008). Moreover, elevated expression of PRMT5 inMelanoma, Breast and Colorectal cancers has been demonstrated tocorrelate with a poor prognosis.

Lymphoid malignancies including CLL are associated with over-expressionof PRMT5. PRMT5 is over-expressed (at the protein level) in the nucleusand cytosol in a number of patient derived Burkitt's lymphoma; mantlecell lymphoma (MCL); in vitro EBV-transformed lymphoma; leukaemia celllines; and B-CLL cell lines, relative to normal CD19+ B lymphocytes (Palet al., 2007; Wang et al., 2008). Intriguingly, despite elevated levelsof PRMT5 protein in these tumour cells, the levels of PRMT5 mRNA arereduced (by a factor of 2-5). Translation of PRMT5 mRNA is however,enhanced in lymphoma cells, resulting in increased levels of PRMT5 (Palet al., 2007; Wang et al., 2008).

In addition to genomic changes, CLL, like almost all cancers, hasaberrant epigenetic abnormalities characterised by globalhypomethylation and hot-spots of repressive hypermethylation ofpromoters including tumour suppressor genes. While the role ofepigenetics in the origin and progression of CLL remains unclear,epigenetic changes appear to occur early in the disease and specificpatterns of DNA methylation are associated with worse prognosis (Chen etal., 2009; Kanduri et al., 2010). Global symmetric methylation ofhistones H3R8 and H4R3 is increased in transformed lymphoid cell linesand MCL clinical samples (Pal et al., 2007), correlating with theoverexpression of PRMT5 observed in a wide variety of lymphoid cancercell lines and MCL clinical samples.

PRMT5 is therefore a target for the identification of novel cancertherapeutics.

PRMT5 Function and Hemoglobinopathies

Hemoglobin is a major protein in red blood cells and is essential forthe transport of oxygen from the lungs to the tissues. In adult humans,the most common hemoglobin type is a tetramer called hemoglobin A,consisting of two α and two β subunits. In human infants, the hemoglobinmolecule is made up of two α and two γ chains. The gamma chains aregradually replaced by subunits as the infant grows. The developmentalswitch in human β-like globin gene subtype from foetal (γ) to adult (β)that begins at birth heralds the onset of the hemoglobinopathiesβ-thalassemia and sickle cell disease (SCD). In β-thalassemia the adultchains are not produced. In SCD a point mutation in the coding sequencein the β globin gene leads to the production of a protein with alteredpolymerisation properties. The observation that increased adult γ-globingene expression (in the setting of hereditary persistence of foetalhemoglobin (HPFH) mutations) significantly ameliorates the clinicalseverity of β-thalassemia and SCD has prompted the search fortherapeutic strategies to reverse γ-globin gene silencing. To date, thishas been achieved through pharmacological induction, using compoundsthat broadly influence epigenetic modifications, including DNAmethylation and histone deacetylation. The development of more targetedtherapies is dependent on the identification of the molecular mechanismsunderpinning foetal globin gene silencing. These mechanisms haveremained elusive, despite exhaustive study of the HPFH mutations, andconsiderable progress in many other aspects of globin gene regulation.

PRMT5 plays a critical role in triggering coordinated repressiveepigenetic events that initiate with dimethylation of histone H4Arginine 3 (H4R3me2s), and culminate in DNA methylation andtranscriptional silencing of the γ-genes (Rank et al., 2010). Integralto the synchronous establishment of the repressive markers is theassembly of a PRMT5-dependent complex containing the DNAmethyltransferase DNMT3A, and other repressor proteins (Rank et al.,2010). DNMT3A is directly recruited to bind to the PRMT5-inducedH4R3me2s mark, and loss of this mark through shRNA-mediated knock-downof PRMT5, or enforced expression of a mutant form of PRMT5 lackingmethyltransferase activity leads to marked upregulation of γ-geneexpression, and complete abrogation of DNA methylation at theγ-promoter. Treatment of human erythroid progenitors with non-specificmethyltransferase inhibitors (Adox and MTA) also resulted inupregulation of γ-gene expression (He Y, 2013). Inhibitors of PRMT5 thushave potential as therapeutics for hemoglobinopathies such asβ-thalassemia and Sickle Cell Disease (SCD).

The present inventors have developed particular substituted β-hydroxyamides inhibit the activity of PRMT5 and therefore may be of use intreating conditions ameliorated by the inhibition of the activity ofPRMT5.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a compound of formulaIa, Ib, Ic or Id:

wherein:n is 1 or 2;

R^(N) is H or Me;

R¹ is optionally one or more halo or methyl groups;R^(2a) and R^(2b) are independently selected from the group consistingof:

-   -   (i) F;    -   (ii) H;    -   (iii) Me; and    -   (iv) CH₂OH;        R^(2c) and R^(2d) (if present) are independently selected from        the group consisting of:    -   (i) F;    -   (ii) H;    -   (iii) Me; and    -   (iv) CH₂OH;        R^(3a) and R^(3b) are independently selected from H and Me;        R^(4a) is selected from OH, —NH₂, —C(═O)NH₂, and —CH₂OH;        R^(4b) is either H or Me;        R⁵ is either H or Me;        m is 1 or 2;        q is 0 or 1;        R^(11a), R^(11b), R^(11c) and R^(11d) are independently selected        from H, halo, C₁₋₄ alkoxy, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄        cycloalkyl, NH—C₁₋₄ alkyl or cyano;        R^(12a) and R^(12b) are independently selected from the group        consisting of:    -   (i) F;    -   (ii) H;    -   (iii) Me; and    -   (iv) CH₂OH;        R^(12c) and R^(12d) are independently selected from the group        consisting of:    -   (i) F;    -   (ii) H;    -   (iii) Me; and    -   (iv) CH₂OH;

R^(12e) is H or Me;

R^(13a) and R^(13b) are independently selected from H and Me;R¹⁴ is either H or Me;R^(16a) and R^(16b) are independently selected from H and Me;R⁶ is selected from H, OMe, and OEt; ora compound of formula:

A second aspect of the present invention provides a compound of thefirst aspect for use in a method of therapy. The second aspect alsoprovides a pharmaceutical composition comprising a compound of the firstaspect and a pharmaceutically acceptable excipient.

A third aspect of the present invention provides a method of treatmentof cancer, comprising administering to a patient in need of treatment, acompound of the first aspect of the invention or a pharmaceuticalcomposition of the first aspect of the invention. The third aspect ofthe present invention also provides the use of a compound of the firstaspect of the invention in the manufacture of a medicament for treatingcancer, and a compound of the first aspect of the invention orpharmaceutical composition thereof for use in the treatment of cancer.

As described below, the compound of the first aspect may be administeredsimultaneously or sequentially with radiotherapy and/or chemotherapy inthe treatment of cancer.

A fourth aspect of the present invention provides a method of treatmentof hemoglobinopathies, comprising administering to a patient in need oftreatment, a compound of the first aspect of the invention or apharmaceutical composition of the first aspect of the invention. Thefourth aspect of the present invention also provides the use of acompound of the first aspect of the invention in the manufacture of amedicament for treating hemoglobinopathies, and a compound of the firstaspect of the invention or pharmaceutical composition of the firstaspect of the invention for use in the treatment of hemoglobinopathies.

Definitions

Halo: The term “halo” as used herein, refers to a group selected fromfluoro, chloro, bromo and iodo.

C₁₋₄ alkyl: The term “C₁₋₄ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a saturated hydrocarbon compound having from 1 to 4 carbonatoms.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), and butyl (C₄).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), and n-butyl (C₄).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄) and tert-butyl (C₄).

C₁₋₄ fluoroalkyl: The term “C₁₋₄ fluoroalkyl” refers to a C₁₋₄ alkylgroup as defined above where one of more of the hydrogen atoms isreplaced by a fluoro. Examples of C₁₋₄ fluoroalkyl include, but are notlimited to, —CF₃, CF₂H, —C₂F₅, and —C₂F₄H.

C₃₋₄ cycloalkyl: the term ‘C₃₋₄ cycloalkyl’ as used herein, pertains toa monovalent moiety obtained by removing a hydrogen atom from a carbonatom of a saturated cyclic core having 3 or 4 atoms in the cyclic coreall of which are carbon atoms. Examples of C₃₋₄ cycloalkyl includecyclopropyl and cyclobutyl.

C₁₋₄ alkoxy: the term “C₁₋₄ alkoxy” as used herein, pertains to a group—OP, where P is C₁₋₄ alkyl as defined above.

Cyano: the term ‘cyano’ as used herein, pertains to a moiety —CN.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO), a salt or solvate thereof, as well asconventional protected forms. Similarly, a reference to an amino groupincludes the protonated form (−N+HR¹R²), a salt or solvate of the aminogroup, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O), a salt or solvate thereof, aswell as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earthcations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examplesof suitable organic cations include, but are not limited to, ammoniumion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺,NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions arethose derived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acidand valeric. Examples of suitable polymeric organic anions include, butare not limited to, those derived from the following polymeric acids:tannic acid, carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Isomers

Certain compounds of the invention may exist in one or more particulargeometric, optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand l or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or l meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, and chlorine, such as, but not limited to ²H(deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S,³⁶Cl, and ¹²⁵I. Various isotopically labeled compounds of the presentinvention, for example those into which radioactive isotopes such as 3H,13C, and 14C are incorporated. Such isotopically labelled compounds maybe useful in metabolic studies, reaction kinetic studies, detection orimaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. Deuterium labelled or substituted therapeutic compounds of theinvention may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism, and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements. An18F labeled compound may be useful for PET or SPECT studies.Isotopically labeled compounds of this invention and prodrugs thereofcan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. Further, substitution with heavierisotopes, particularly deuterium (i.e., 2H or D) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent. The concentration of such aheavier isotope, specifically deuterium, may be defined by an isotopicenrichment factor. In the compounds of this invention any atom notspecifically designated as a particular isotope is meant to representany stable isotope of that atom.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Therapeutic Indications

Compounds disclosed herein may provide a therapeutic benefit in a numberof disorders, in particular, in the treatment or prevention of cancersand hemoglobinopathies.

Cancer

Modulators of PRMT5 mediated post-translational arginine methylation ofp53 may regulate a pro-apoptotic p53 response, and may therefore beuseful as therapeutic agents, for example in the treatment of cancer.Such agents may also be useful as therapeutic agents for the treatmentof cancers which exhibit overexpression of PRMT5.

A “cancer” may be any form of cancer. In particular, a cancer cancomprise any one or more of the following: leukemia, acute lymphocyticleukemia (ALL), acute myeloid leukemia (AML), chronic lymphocyticleukemia (CLL), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma,Hodgkin's disease, prostate cancer, lung cancer, melanoma, breastcancer, colon and rectal cancer, colon cancer, squamous cell carcinomaand gastric cancer.

Alternatively, the cancer may comprise adrenocortical cancer, analcancer, bladder cancer, blood cancer, bone cancer, brain tumor, cancerof the female genital system, cancer of the male genital system, centralnervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma,childhood sarcoma, endometrial cancer, endometrial sarcoma, esophagealcancer, eye cancer, gallbladder cancer, gastrointestinal tract cancer,hairy cell leukemia, head and neck cancer, hepatocellular cancer,hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngealcancer, liver cancer, malignant fibrous histiocytoma, malignant thymoma,mesothelioma, multiple myeloma, myeloma, nasal cavity and paranasalsinus cancer, nasopharyngeal cancer, nervous system cancer,neuroblastoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma,ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer,pharyngeal cancer, pituitary tumor, plasma cell neoplasm, primary CNSlymphoma, rectal cancer, respiratory system, retinoblastoma, salivarygland cancer, skin cancer, small intestine cancer, soft tissue sarcoma,stomach cancer, stomach cancer, testicular cancer, thyroid cancer,urinary system cancer, uterine sarcoma, vaginal cancer, vascular system,Waldenstrom's macroglobulinemia and/or Wilms' tumor. Cancers may be of aparticular type. Examples of types of cancer include lymphoma, melanoma,carcinoma (e.g. adenocarcinoma, hepatocellular carcinoma, medullarycarcinoma, papillary carcinoma, squamous cell carcinoma), astrocytoma,glioma, medulloblastoma, myeloma, meningioma, neuroblastoma, sarcoma(e.g. angiosarcoma, chrondrosarcoma, osteosarcoma).

The cancer may be a PRMT5 overexpressing cancer. The cancer may overexpress PRMT5 protein relative to non-cancerous tissue. In some cases,the cancer overproduces PRMT5 mRNA relative to non-cancerous tissue.

Alternatively or additionally, the cancer may be a p53 overexpressingcancer. The cell may overexpress p53 protein relative to non-canceroustissue. It may overproduce p53 mRNA as compared to non-cancerous tissue.In some cases, the level of p53 protein and/or mRNA in the cell is at alevel approximately equivalent to that of a non-cancerous cell.

The agents described herein may be useful in combination with otheranti-cancer therapies. They may act synergistically with chemo- orradiotherapy, and/or with p53 targeted drugs.

An inhibitor of PRMT5 would in all likelihood augment the effects ofdrugs (such as the nutlins) that restore p53. Inhibition of PRMT5,resulting in decreased arginine-methylated p53, may sensitize tumourcells to chemo- and radiotherapy by switching, or at least biasing, thecellular outcome to apoptosis.

Combination Therapies

p53 is activated by DNA damage. PRMT5 is part of the complex of proteinsthat activate and modulate p53 activity in response to DNA damage. It islikely that inhibition of PRMT5, resulting in decreasedarginine-methylated p53, would sensitize tumour cells to chemo- andradiotherapy by switching or at least biasing the cellular outcome toapoptosis. PRMT5 inhibition is likely to synergize well with low dosechemo- or radiotherapy, by stabilizing p53, and biasing the cellularoutcome to apoptosis.

Biasing the p53 response towards apoptosis would in all likelihood be ofbenefit, and an agent that so biases the response would be expected toaugment the effect of a p53 resurrecting drug. Thus, in some cases, aPRMT5 modulator disclosed herein may be administered in conjunction witha radiotherapeutic or chemotherapeutic regime. It may be administered inconjunction with a drug that resurrects cellular p53 activity, forexample, a p53 agonist. The PRMT5 modulator may be administeredsimultaneously or sequentially with radio and/or chemotherapy. Suitablechemotherapeutic agents and radiotherapy protocols will be readilyappreciable to the skilled person. In particular, the compound describedherein may be combined with low dose chemo or radio therapy. Appropriatedosages for “low dose” chemo or radio therapy will be readilyappreciable to the skilled practitioner.

Hemoglobinopathies

The compounds disclosed herein may be useful in the treatment orprevention of conditions that may benefit from the increased expressionof γ-globin genes, for example, due to the release of repressivemethylation of these genes. The compounds disclosed herein may be usefulin the treatment or prevention of hemoglobinopathies. A hemoglobinopathyis a condition associated with the presence of abnormal hemoglobin inthe blood of a subject. Such conditions include β-thalassemia and SickleCell Disease, α-thalassemia and δ-thalassemia.

Hemoglobinopathies treatable by the compounds disclosed herein may beameliorated by the re-activation of the subjects γ-globin genes (γgenes). In such cases, the subject is not a fetal mammal.

Methods of Treatment

The compounds of the present invention may be used in a method oftherapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a compound of the invention. Theterm “therapeutically effective amount” is an amount sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage, is within the responsibility of general practitioners and othermedical doctors.

As described above, the anti cancer treatment defined herein may beapplied as a sole therapy or may involve, in addition to the compound ofthe invention, conventional surgery or radiotherapy or chemotherapy.Such chemotherapy may include one or more of the following categories ofanti-tumour agents:—

(i) other antiproliferative/antineoplastic drugs and combinationsthereof, as used in medical oncology, such as alkylating agents (forexample cisplatin, oxaliplatin, carboplatin, cyclophosphamide, nitrogenmustard, melphalan, chlorambucil, busulphan, temozolamide andnitrosoureas); antimetabolites (for example gemcitabine and antifolatessuch as fluoropyrimidines like 5 fluorouracil and tegafur, raltitrexed,methotrexate, cytosine arabinoside, and hydroxyurea); antitumourantibiotics (for example anthracyclines like adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and docetaxel (Taxotere) and polokinase inhibitors);and topoisomerase inhibitors (for example epipodophyllotoxins likeetoposide and teniposide, amsacrine, topotecan and camptothecin);(ii) cytostatic agents such as antioestrogens (for example tamoxifen,fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene),antiandrogens (for example bicalutamide, flutamide, nilutamide andcyproterone acetate), LHRH antagonists or LHRH agonists (for examplegoserelin, leuprorelin and buserelin), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example as anastrozole,letrozole, vorazole and exemestane) and inhibitors of 5*-reductase suchas finasteride;(iii) anti-invasion agents (for example c-Src kinase family inhibitorslike4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4-yloxyquinazoline(AZD0530; International Patent Application WO 01/94341),N-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-ylamino}thiazole-5-carboxamide(dasatinib, BMS-354825; J. Med. Chem., 2004, 47, 6658-6661 and4-((2,4-dichloro-5-methoxyphenyl)amino)-6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinoline-3-carbonitrile(bosutinib, SKI-606; Cancer research (2003), 63(2), 375-81), andmetalloproteinase inhibitors like marimastat, inhibitors of urokinaseplasminogen activator receptor function or antibodies to Heparanase);(iv) inhibitors of growth factor function: for example such inhibitorsinclude growth factor antibodies and growth factor receptor antibodies(for example the anti erbB2 antibody trastuzumab [HerceptinT], theanti-EGFR antibody panitumumab, the anti erbB1 antibody cetuximab[Erbitux, C225] and any growth factor or growth factor receptorantibodies disclosed by Stern et al. Critical reviews inoncology/haematology, 2005, Vol. 54, pp 11-29); such inhibitors alsoinclude tyrosine kinase inhibitors, for example inhibitors of theepidermal growth factor family (for example EGFR family tyrosine kinaseinhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, ZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI 774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine(CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib,inhibitors of the hepatocyte growth factor family, inhibitors of theplatelet-derived growth factor family such as imatinib, inhibitors ofserine/threonine kinases (for example Ras/Raf signalling inhibitors suchas farnesyl transferase inhibitors, for example sorafenib (BAY43-9006)), inhibitors of cell signalling through MEK and/or AKT kinases,inhibitors of the hepatocyte growth factor family, c-kit inhibitors, ablkinase inhibitors, IGF receptor (insulin-like growth factor) kinaseinhibitors; aurora kinase inhibitors (for example AZD1152, PH739358,VX-680, MLN8054, R763, MP235, MP529, VX-528 AND AX39459) and cyclindependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors;(v) antiangiogenic and antilymphangiogenic agents such as those whichinhibit the effects of vascular endothelial growth factor, [for examplethe anti vascular endothelial cell growth factor A (VEGFA) antibodybevacizumab (AvastinT), the anti vascular endothelial cell growth factorA (VEGFA) antibody ranibizumab, the anti-VEGF aptamer pegaptanib, theanti vascular endothelial growth factor receptor 3 (VEGFR3) antibodyIMC-3C₅, the anti vascular endothelial cell growth factor C (VEGFC)antibody VGX-100, the anti vascular endothelial cell growth factor D(VEGFD) antibody VGX-200, the soluble form of the vascular endothelialgrowth factor receptor 3 (VEGFR3) VGX-300 and VEGF receptor tyrosinekinase inhibitors such as4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(vandetanib; ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(cediranib; AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787;WO 98/35985), pazopanib (GW786034), axitinib (AG013736), sorafenib andsunitinib (SU11248; WO 01/60814), compounds such as those disclosed inInternational Patent Applications WO97/22596, WO 97/30035, WO 97/32856and WO 98/13354 and compounds that work by other mechanisms (for examplelinomide, inhibitors of integrin avb3 function and angiostatin)];(vi) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, WO 00/40529,WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;(vii) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense;(viii) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2,GDEPT (gene directed enzyme pro drug therapy) approaches such as thoseusing cytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi drug resistance gene therapy; and(ix) immunotherapy approaches, including for example ex vivo and in vivoapproaches to increase the immunogenicity of patient tumour cells, suchas transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte macrophage colony stimulating factor, approaches to decreaseT cell anergy, approaches using transfected immune cells such ascytokine transfected dendritic cells, approaches using cytokinetransfected tumour cell lines and approaches using anti idiotypicantibodies

Administration

The active compound or pharmaceutical composition comprising the activecompound may be administered to a subject by any convenient route ofadministration, whether systemically/peripherally or at the site ofdesired action, including but not limited to, oral (e.g. by ingestion);topical (including e.g. transdermal, intranasal, ocular, buccal, andsublingual); pulmonary (e.g. by inhalation or insufflation therapyusing, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal;parenteral, for example, by injection, including subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intracardiac,intrathecal, intraspinal, intracapsular, subcapsular, intraorbital,intraperitoneal, intratracheal, subcuticular, intraarticular,subarachnoid, intravitreal and intrasternal; by implant of a depot, forexample, subcutaneously, intravitreal or intramuscularly. The subjectmay be a eukaryote, an animal, a vertebrate animal, a mammal, a rodent(e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse),canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), aprimate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset,baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or ahuman.

Formulations

While it is possible for the active compound to be administered alone,it is preferable to present it as a pharmaceutical composition (e.g.formulation) comprising at least one active compound, as defined above,together with one or more pharmaceutically acceptable carriers,adjuvants, excipients, diluents, fillers, buffers, stabilisers,preservatives, lubricants, or other materials well known to thoseskilled in the art and optionally other therapeutic or prophylacticagents.

Thus, the present invention further provides pharmaceuticalcompositions, as defined above, and methods of making a pharmaceuticalcomposition comprising admixing at least one active compound, as definedabove, together with one or more pharmaceutically acceptable carriers,excipients, buffers, adjuvants, stabilisers, or other materials, asdescribed herein.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g. human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

Suitable carriers, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing into association the activecompound with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

Formulations may be in the form of liquids, solutions, suspensions,emulsions, elixirs, syrups, tablets, losenges, granules, powders,capsules, cachets, pills, ampoules, suppositories, pessaries, ointments,gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses,electuaries, or aerosols.

Formulations suitable for oral administration (e.g. by ingestion) may bepresented as discrete units such as capsules, cachets or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or suspension in an aqueous or non-aqueousliquid; or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion; as a bolus; as an electuary; or as a paste.

A tablet may be made by conventional means, e.g., compression ormoulding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine the activecompound in a free-flowing form such as a powder or granules, optionallymixed with one or more binders (e.g. povidone, gelatin, acacia,sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers ordiluents (e.g. lactose, microcrystalline cellulose, calcium hydrogenphosphate); lubricants (e.g. magnesium stearate, talc, silica);disintegrants (e.g. sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose); surface-active ordispersing or wetting agents (e.g. sodium lauryl sulfate); andpreservatives (e.g. methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,sorbic acid). Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activecompound therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile. Tablets mayoptionally be provided with an enteric coating, to provide release inparts of the gut other than the stomach.

Formulations suitable for topical administration (e.g. transdermal,intranasal, ocular, buccal, and sublingual) may be formulated as anointment, cream, suspension, lotion, powder, solution, past, gel, spray,aerosol, or oil. Alternatively, a formulation may comprise a patch or adressing such as a bandage or adhesive plaster impregnated with activecompounds and optionally one or more excipients or diluents.

Formulations suitable for topical administration in the mouth includelosenges comprising the active compound in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activecompound in an inert basis such as gelatin and glycerin, or sucrose andacacia; and mouthwashes comprising the active compound in a suitableliquid carrier.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active compound is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the active compound.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of about 20 to about 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid for administrationas, for example, nasal spray, nasal drops, or by aerosol administrationby nebuliser, include aqueous or oily solutions of the active compound.

Formulations suitable for administration by inhalation include thosepresented as an aerosol spray from a pressurised pack, with the use of asuitable propellant, such as dichlorodifluoromethane,trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, orother suitable gases.

Formulations suitable for topical administration via the skin includeointments, creams, and emulsions. When formulated in an ointment, theactive compound may optionally be employed with either a paraffinic or awater-miscible ointment base. Alternatively, the active compounds may beformulated in a cream with an oil-in-water cream base. If desired, theaqueous phase of the cream base may include, for example, at least about30% w/w of a polyhydric alcohol, i.e., an alcohol having two or morehydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol,sorbitol, glycerol and polyethylene glycol and mixtures thereof. Thetopical formulations may desirably include a compound which enhancesabsorption or penetration of the active compound through the skin orother affected areas. Examples of such dermal penetration enhancersinclude dimethylsulfoxide and related analogues.

When formulated as a topical emulsion, the oily phase may optionallycomprise merely an emulsifier (otherwise known as an emulgent), or itmay comprises a mixture of at least one emulsifier with a fat or an oilor with both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabiliser. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabiliser(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulphate. The choice of suitable oils or fats for the formulationis based on achieving the desired cosmetic properties, since thesolubility of the active compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required.

Alternatively, high melting point lipids such as white soft paraffinand/or liquid paraffin or other mineral oils can be used.

Formulations suitable for rectal administration may be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active compound, such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration (e.g. by injection,including cutaneous, subcutaneous, intramuscular, intravenous andintradermal), include aqueous and non-aqueous isotonic, pyrogen-free,sterile injection solutions which may contain anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. Examples of suitable isotonic vehicles for use insuch formulations include Sodium Chloride Injection, Ringer's Solution,or Lactated Ringer's Injection. Typically, the concentration of theactive compound in the solution is from about 1 ng/mL to about 10 μg/mL,for example from about 10 ng/ml to about 1 μg/mL. The formulations maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets. Formulations may be in the form ofliposomes or other microparticulate systems which are designed to targetthe active compound to blood components or one or more organs.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the compound, and compositions comprising the compound, canvary from patient to patient. Determining the optimal dosage willgenerally involve the balancing of the level of therapeutic benefitagainst any risk or deleterious side effects. The selected dosage levelwill depend on a variety of factors including, but not limited to, theactivity of the particular compound, the route of administration, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds, and/or materials usedin combination, the severity of the condition, and the species, sex,age, weight, condition, general health, and prior medical history of thepatient. The amount of compound and route of administration willultimately be at the discretion of the physician, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action which achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range ofabout 100 ng to about 25 mg (more typically about 1 μg to about 10 mg)per kilogram body weight of the subject per day. Where the activecompound is a salt, an ester, an amide, a prodrug, or the like, theamount administered is calculated on the basis of the parent compoundand so the actual weight to be used is increased proportionately.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 100 mg, 3 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 150 mg, 2 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 200 mg, 2 timesdaily.

However in one embodiment, the active compound is administered to ahuman patient according to the following dosage regime: about 50 orabout 75 mg, 3 or 4 times daily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 100 or about 125mg, 2 times daily.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, regression of the condition,amelioration of the condition, and cure of the condition. Treatment as aprophylactic measure (i.e., prophylaxis, prevention) is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosagefrom comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio, when administered in accordance with a desiredtreatment regimen.

Similarly, the term “prophylactically-effective amount,” as used herein,pertains to that amount of an active compound, or a material,composition or dosage from comprising an active compound, which iseffective for producing some desired prophylactic effect, commensuratewith a reasonable benefit/risk ratio, when administered in accordancewith a desired treatment regimen.

The Subject/Patient

The subject/patient may be an animal, mammal, a placental mammal, amarsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilledplatypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse),murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., abird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., ahorse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., acow), a primate, simian (e.g., a monkey or ape), a monkey (e.g.,marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang,gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus. In one preferred embodiment, the subject/patientis a human.

General Synthesis Methods

The compounds of the invention can be prepared employing the followinggeneral methods and using procedures described in detail in theexamples. The reaction conditions referred to are illustrative andnon-limiting, for example one skilled in the art may use a diverse rangeof synthetic methods to synthesis the desired compounds such as but notlimited to methods described in literature (for example but not limitedto March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 7th Edition or Larock's Comprehensive OrganicTransformations: Comprehensive Organic Transformations: A Guide toFunctional Group Preparations).

Compounds of formulae Ia, Ib and Ic

Compounds of formulae Ia, Ib and Ic, as described above, can be preparedby synthetic strategies outlined below, wherein the definitions aboveapply. The synthetic strategies could be applied to the use of racemicor single enantiomer starting materials.

General Synthesis Method 1

Scheme 1A illustrates the formation of the amide bond by coupling arelevant carboxylic acid to a primary amine or a secondary amine G1.Methods to form such amides will be apparent to those skilled in theart, but include for example the use of reagents such as HATU, HBTU, T3Pand EDCl/HOBt, and the use of activated forms of the carboxylic acidsuch as the corresponding acyl halide, mixed anhydride orN-hydroxysuccinimide ester.

R⁷ represents the fused cyclic system.

Optionally, where R^(N)=H, a protecting group (PG) may be used such asbut not limited to Boc, allyl, benzyl, 2,4-dimethoxybenzyl. Asillustrated in Scheme 1B, coupling between a relevant carboxylic acid oractivated acid, e.g. acyl halide to a primary amine or a secondary amineG3 bearing a protecting group (PG) is performed as described previously.Conditions for the removal of the protecting group are dependent on thetype of protecting group employed, and may include but is not limited tosuch methods as acid hydrolysis, transition metal catalysed cleavage andhydrogenation over transition metal catalysts. Other suitable protectinggroups and removal methods will be known to those skilled in the art(for example Greene's Protective Groups in Organic Synthesis, 4thEdition). The use of such a protecting group could be relevant in theother Schemes described.

Where A contains a direct carboxylic acid substitution or a furthersubstitution that also has a carboxylic acid substitution G5, anotheramide formation can be conducted to provide compounds of G6, scheme 1C.

General Synthesis Method 2

Scheme 2A illustrates the synthesis of the substituted amine alcohol G9.This is achieved by opening the epoxide with a desired amine (HNR^(N)R⁷)to form the intermediate G7. The phthalimide protecting group can thenbe removed by heating with hydrazine hydrate to form G8. Other suitableprotecting groups and removal methods will be known to those skilled inthe art (for example Greene's Protective Groups in Organic Synthesis,4th Edition). Commercially available enantiomerically pure epoxidescould be used to give single enantiomer products G9.

The amide formation to form G9 can be achieved by the methods outline inScheme 1A. The synthesis of either enantiomer and the racemate can beachieved by the same method.

General Synthesis Method 3

Where R^(7a) and R^(7b) together with the atom to which they are boundform R⁷.

Scheme 3A illustrates the synthesis of compounds with the formula G13,beginning with an amide bond formation by coupling a relevant carboxylicacid to a primary amine or a secondary amine G10. Methods to form suchamides will be apparent to those skilled in the art, but include forexample the use of reagents such as HATU, HBTU, T3P and EDCl/HOBt, andthe use of activated forms of the carboxylic acid such as thecorresponding acyl halide, mixed anhydride or N-hydroxysuccinimideester.

Suitably protected amino groups and methods for the removal of saidprotecting groups will be known to those skilled in the art (for exampleGreene's Protective Groups in Organic Synthesis, 4th Edition). Suitableprotecting groups might include Boc, CBz, or phthalimide. Upon removalof the protecting group to provide compounds of the general formula G12,these intermediates can be converted to the desired compound, G13.Methods to form such an intermediate will be apparent to those skilledin the art, but include for example reductive amination by the use ofreagents such as, but not limited to sodium triacetoxyborohydride orsodium cyanoborohydride with acetic acid, and sodiumtriacetoxyborohydride triacetoxyborohydride or sodium borohydride withTi(O^(i)Pr)₄.

General Synthesis Method 4

Scheme 4A illustrates an approach to the synthesis of compounds with theformula of G16, beginning with the reaction of the desired amine(HNR^(N)R⁷) with an epoxide bearing a suitable protected amino groupsuch as for example tert-butyl N-(oxiran-2-yl methyl) carbamate. Openingof the epoxide under suitable conditions furnishes the intermediatecompound G14. Suitably protected amino groups and methods for theremoval of said protecting groups will be known to those skilled in theart (for example Greene's Protective Groups in Organic Synthesis, 4thEdition). Upon removal of the protecting group to provide compounds ofthe general formula G15, these intermediates can be converted to thedesired compound, G16, by the procedure outlined in Scheme 1A.

General Synthesis Method 5

Scheme 5A illustrates how to form amine substitutions such as shown inG17. An amide is reacted with a halo-epoxide or similar and theresultant mixture reacted on with a desired amine (HNR⁶R⁷) resulting ina product within the scope of G17. The group denoted (X) can be but isnot limited to halogen, tosylate, nosylate or similar.

Alternatively enantiomerically pure forms of the epoxide, e.g.epichlorohydrin, can be used to obtain the products G17 inenantiomerically pure form.

General Synthesis Method 6

Scheme 6A illustrates how to form amine substitutions such as shown inG19. Suitable amide protecting groups and methods for the removal ofsaid protecting groups will be known to those skilled in the art (forexample Greene's Protective Groups in Organic Synthesis, 4th Edition).An amide is reacted with a halo-epoxide or similar and the resultantmixture reacted on with a desired amine (HNR^(N)R⁷) resulting in anintermediate G18. The group denoted (X) can be but is not limited tohalogen, tosylate, nosylate or similar. Removal of the protecting groupprovides compounds of the general formula G19

Alternatively enantiomerically pure forms of the epoxide for exampleinclude but are not limited to epichlorohydrin, can be used to obtainthe products G14 in enantiomerically pure form.

General Synthesis Method 7

Scheme 7A illustrates an approach to the synthesis of compounds with theformula of G23, beginning with the reaction of the desired amine(HNR^(N)R⁷) with an epoxide bearing a leaving group (LG). The grouprepresented by (LG) includes but not limited to halide, mesylate,tosylate, nosylate. Depending on the conditions and the nature of theleaving group, the reaction can proceed through two different pathwaysto give either epoxide with structure G20 or alcohol with structure G21.Treatment of either compound with primary amine (R⁵—NH₂) furnishesintermediates with structure G22. These intermediates can be convertedto the desired compound, G23, by the procedure outlined in Scheme 1A.

General Synthesis Method 8

Scheme 8A illustrates the synthesis of compounds G27 which begins with acoupling of a compound G24 with a desired amine (HNR^(N)R⁷) bysubstitution of a leaving group (LG) to give intermediates G25. Thegroup represented by (LG) includes but is not limited to halide,mesylate, tosylate, nosylate. The group represented by (PG) is asuitable amine protecting group which includes but is not limited toBoc, phthalimide, benzyl, PMB, allyl. Suitable amine protecting groupsand methods for the removal of said protecting groups will be known tothose skilled in the art (for example Greene's Protective Groups inOrganic Synthesis, 4th Edition). Upon removal of the protecting group,coupling with a suitable carboxylic acid can be performed by methodsillustrated in Scheme 1A.

Alternatively, as shown in Scheme 8B, compounds with structure G28 canbe coupled with the desired amine (HNR^(N)R⁷) to give compounds G27.

General Synthesis Method 9

Scheme 9A illustrates the synthesis of compounds G32 where R^(3a) andR^(3b) are hydrogen. Amide bond formation of compounds with structureG28 with the desired amine (HNR^(N)R⁷) give compounds with structureG29. Methods to form such amides will be apparent to those skilled inthe art, but include for example the use of reagents such as HATU, HBTU,T3P and EDCl/HOBt, and the use of activated forms of the carboxylic acidsuch as the corresponding acyl halide, mixed anhydride orN-hydroxysuccinimide ester. Reduction of the amide to the correspondingamine G30 will be apparent to those skilled in the art but such methodsinclude but are not limited to the use of a reducing agent such asLiAlH₄. Suitable amine protecting groups and methods for the removal ofsaid protecting groups will be known to those skilled in the art (forexample Greene's Protective Groups in Organic Synthesis, 4th Edition).Upon deprotection, intermediates G31 can be converted to the desiredcompound, G32, by the procedure outlined in Scheme 1A.

Alternatively where X=H in G28, a reductive amination could beperformed, for example using sodium triacetoxy borohydride and aceticacid to form G30 directly, rather than proceeding via intermediate G29.

General Synthesis Method 10

Scheme 10A illustrates the addition of an amine (HNR⁸R⁹) as asubstituent which is a part of A. This can be achieved by coupling arelevant carboxylic acid to a primary amine or a secondary amine,NHR⁸R⁹. Methods to form such amides will be apparent to those skilled inthe art, but include for example the use of reagents such as HATU, HBTU,T3P and EDCl/HOBt, and the use of activated forms of the carboxylic acidsuch as the corresponding acyl halide, mixed anhydride orN-hydroxysuccinimide ester. The group denoted by (X) may be but notlimited to halogen, tosylate or other suitable group. Conversion of (X)in G33 into an ester in G34 will be apparent to those skilled in theart, but include for example a carbonylation reaction which can beachieve by the use of carbon monoxide in the presence of an transitionmetal catalyst such as but not limited to PdCl₂dppf.DCM; and analcoholic solvent such as but not limited to methanol, ethanol,isopropanol or tert-butyl alcohol. Formation of the carboxylic acid canbe achieved by for example hydrolysis with a base such as an alkalimetal hydroxide or an acid for example aqueous hydrochloric acid to formG35. The amide formation to form G36 can be achieved by the methodsoutline in Scheme 1A.

Alternatively, for the synthesis of ester G34 the order of steps can bereversed as described in Scheme 10B.

Alternatively for the synthesis of amide G36 the steps may be reorderedsuch that the formation of the R⁸R⁹N amide on the A substituent occursafter the coupling of A to the amine G26. This may be achieved bycoupling a suitable amine with an intermediate where A bears a suitablefunctional group for coupling, for example but not limited to acarboxylic acid or alkali metal carboxylate salt, as shown in Scheme 10C

Compounds of Formula Id

Compounds of formula Id, as described above, can be prepared bysynthetic strategies outlined below, wherein the definitions aboveapply.

General Synthesis 11

Scheme 11A illustrates the synthesis of compounds with the structureG49. A coupling of a carbonyl compound of structure G41 with anorganometallic compound of structure G42 to give a compound withstructure G43 will be apparent to those skilled in the art. The grouprepresented by (M) includes but is not limited to Mg, In, Zn and thegroup represented by (X) may be a halide where (Y) may be the number1-3. Suitable protected amino groups represented by (PG) include but arenot limited to phthalimide; and methods for the removal of saidprotecting groups will be known to those skilled in the art (for exampleGreene's Protective Groups in Organic Synthesis, 4th Edition). Synthesisof compounds with structure G45 is performed by reacting alkyne G43 withcompounds of structure G44 in the presence of a transition metalcatalyst or combination of transition metal catalysts such as but notlimited to bis(triphenylphosphine)nickel(II) chloride/Zn.

After removal of the protecting group, methods to synthesise amides G48will be apparent to those skilled in the art, but include for examplethe use of reagents such as HATU, HBTU, T3P and EDCl/HOBt, and the useof activated forms of the carboxylic acid G47 such as the correspondingacyl halide, carbamate or N-hydroxysuccinimide ester. Transformation ofisoquinolines of structure G48 to give tetrahydroisoquinolines ofstructure G49 will be apparent to those skilled in the art and suchmethods include but are not limited to reduction in the presence of atransition metal catalyst.

Alternatively, for the synthesis of compounds with structure G52 whereR^(2a), R^(2b), R^(2d)=H, a coupling of an alkyne and a aryl halide willbe apparent to those skilled in the art and such methods include acoupling in the presence of a transition metal catalyst or catalystcombination such as but not limited to PdCl₂(PPh₃)₂/CuI andPd(OAc)₂/PPh₃. This may be followed by cyclisation either in situ or asa separate step to give isoquinoline of structure G51. The syntheticsteps to give compounds with general formula G52 will be similar tothose used in Scheme 11A.

General Synthesis 12

Where R¹⁷ represents the fused ring group.

Scheme 12A illustrates the synthesis of compounds G58 from aldehyde G53(or ketone where R¹⁴=Me). Conversion of a carbonyl to an alkene will beapparent to those skilled in the art but methods include but are notlimited to a Wittig reaction with [Ph₃PMe]⁺Br⁻ in the presence of a basesuch as KHMDS. The alkene G54 can be epoxidised with reagents such asmCPBA and then reacted with an amine to give intermediate G57.Alternatively, an aminohydroxylation can be performed by methods such asbut not limited to reaction with (PG)NHOTs in the presence of potassiumosmate dihydrate to give G56. Removal of the protecting group will beapparent to those skilled in the art (for example Greene's ProtectiveGroups in Organic Synthesis, 4th Edition) and gives intermediate G57.Amide bond formation to give compounds G58 can be performed by methodspreviously described (General synthesis 11).

General Synthesis 13

Scheme 13A illustrates the synthesis of compounds G62 beginning with aHenry reaction between an aldehyde G59 (or ketone where R¹⁴=Me) andnitromethane in the presence or absence of a suitable base, such as butnot limited to DBU, a KF, TBAF or sodium hydroxide, in the presence orabsence of a chiral or achiral transition metal compound for example butnot limited to complexes of copper, cobalt or zinc to furnish anitro-alcohol G60. Reduction of the nitro group to the primary amine G61will be apparent to those skilled in the art and include but are notlimited to using reducing conditions such as a transition metal (Fe, In,Zn) in the presence of HCl, hydrogenation in the presence of atransition metal or transition metal catalyst. Amide bond formation togive compounds G62 can be performed by methods previously described(General synthesis 11). The method can also be carried out withnitroethane and other nitroalkanes, as appropriate.

General Synthesis 14

Scheme 14A illustrates the addition of an amine (HNR⁸R⁹), as asubstituent which is a part of A. This can be achieved by coupling arelevant carboxylic acid to a primary amine or a secondary amine,NHR⁸R⁹. Methods to form such amides will be apparent to those skilled inthe art, but include for example the use of reagents such as HATU, HBTU,T3P and EDCl/HOBt, and the use of activated forms of the carboxylic acidsuch as the corresponding acyl halide, mixed anhydride orN-hydroxysuccinimide ester. The group denoted by (X) may be but notlimited to halogen, tosylate or other suitable group. Conversion of (X)in G62 into an ester in G63 will be apparent to those skilled in theart, but include for example a carbonylation reaction which can beachieved by the use of carbon monoxide in the presence of an transitionmetal catalyst such as but not limited to PdCl₂dppf.DCM; and analcoholic solvent such as but not limited to methanol, ethanol,isopropanol or tert-butyl alcohol. Formation of the carboxylic acid canbe achieved by for example hydrolysis with a base such as an alkalimetal hydroxide or an acid for example aqueous hydrochloric acid to formG64. The amide formation to form G65 can be achieved by the methodsoutline in Scheme 11A.

Alternatively, for the synthesis of ester G63 the order of steps can bereversed as described in Scheme 14B.

Alternatively for the synthesis of amide G65 the steps may be reorderedsuch that the formation of the R⁸R⁹N amide on the A substituent occursafter the coupling of A to the primary amine G61. This may be achievedby coupling a suitable amine with an intermediate where A bears asuitable functional group for coupling, for example but not limited to acarboxylic acid or alkali metal carboxylate salt, as shown in Scheme14C.

FURTHER EMBODIMENTS

n

In some embodiments, n is 1. In some embodiments, n is 2.

R^(N)

In some embodiment, R^(N) is H. In other embodiments, R^(N) is Me.

R¹

In some embodiments, there may be no R¹ substituents. In someembodiments, R¹ represents one to four Me or halo groups, preferably oneto three Me or halo groups and more preferably one or two Me or halogroups. In some of these embodiments, R¹ may represent F. In others ofthese embodiments, R¹ may represent Me groups.

R²

R^(2a), R^(2b), R^(2c) and R^(2d) (if present) are independentlyselected from H, F, CH₂OH and Me. In some of these embodiments, R^(2a),R^(2b), R^(2c) and R^(2d) (if present) are independently selected fromH, Me and CH₂OH. In further of these embodiments, R^(2a), R^(2b), R^(2c)and R^(2d) (if present) are independently selected from H and Me.

In some embodiments R^(2a), R^(2b), R^(2c) and R^(2d) (if present) areall H.

In some of those embodiments where R^(2a), R^(2b), R^(2c) and R^(2d) areall present (i.e. formulae Ia and Ib), they may be comprised of three Hand one Me or CH₂OH group. It may be preferred in these embodiments thatR^(2a) is Me and R^(2b), R^(2c) and R^(2d) are H. It may be preferred inthese embodiments that R^(2c) is Me or CH₂OH and R^(2a), R^(2b) andR^(2d) are H.

In some of those embodiments where R^(2a), R^(2b), R^(2c) and R^(2d) areall present (i.e. formulae Ia and Ib), they may be comprised of two Hand two Me groups. It may be preferred in these embodiments that R^(2a)and R^(2c) are Me and R^(2b) and R^(2d) are H. It may be preferred inthese embodiments that R^(2a) and R^(2b) are Me and R^(2c) and R^(2d)are H. It may also be preferred in these embodiments that R^(2c) andR^(2d) are Me and R^(2a) and R^(2b) are H.

In some of those embodiment where only R^(2a) and R^(2b) are present(i.e. formula Ic), they may be comprised of one H and one Me or CH₂OHgroup.

In some of those embodiment where only R^(2a) and R^(2b) are present(i.e. formula Ic), they may both be a Me or CH₂OH group.

R³

R^(3a) and R^(3b) are independently selected from H and Me. In someembodiments R^(3a) is H and R^(3b) is Me. In some embodiments R^(3a) andR^(3b) are both H. In some embodiments R^(3a) and R^(3b) are both Me.

R^(4a)

In some embodiments R^(4a) is OH.

In other embodiments, R^(4a) is —NH₂. In other embodiments R^(4a) is—C(═O)NH₂. In other embodiments R^(4a) is —CH₂OH.

It may be preferred that R^(4a) is OH.

R^(4b)

In some embodiments R^(4b) is H. In some embodiments R^(4b) is Me.

R⁵

In some embodiments R⁵ is H. In some embodiments R⁵ is Me.

Enantiomers

The carbon to which R^(4a) and R^(4b) are attached is a chiral centre.

When the compound contains this chiral centre, in some embodiments, thecompound is a racemate.

When the compound contains this chiral centre, in some embodiments, thecompound is a single enantiomer. In some of these embodiments, thecompound is the (R)-enantiomer. In others of these embodiments, thecompound is the (S)-enantiomer.

The compound may also include further chiral centres, for example, incompounds of formula Ia, in tetrahydronaphthalenyl group. In someembodiments, the compound is a racemate. In other embodiments, thecompound is a single enantiomer. In some of these embodiments, thecompound is the (R)-enantiomer. In others of these embodiments, thecompound is the (S)-enantiomer.

R¹-R⁵ & n

In some embodiments, R^(N), R¹, R^(2a), R^(2b), R^(2c) (if present),R^(2d) (if present), R^(3a), R^(3b), R^(4b) and R⁵ are all H, R^(4a) isOH and n is 1, and thus the compound of formula Ia is of formula Ia1,the compound of formula Ib is of formula Ib1, and the compound offormula Ic is of formula Id:

In some embodiments, R¹, R^(2a), R^(2b), R^(2c) (if present), R^(2d) (ifpresent), R^(3a), R^(3b), R^(4b) and R⁵ are all H, R^(4a) is OH and n is2, and thus the compound of formula Ia is of formula Ia2, the compoundof formula Ib is of formula Ib2, and the compound of formula Ic is offormula Ic2:

m

In some embodiments, m is 1. In some embodiments, m is 2.

q

In some embodiments, q is 0. In some embodiments, q is 1.

R^(11a), R^(11b), R^(11c) and R^(11d)

In some embodiments, R^(11a), R^(11b), R^(11c) and R^(11d) are all H.

In some embodiments, one of R^(11a), R^(11b), R^(11c) and R^(11d) isC₁₋₄ alkoxy, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄ cycloalkyl, NH—C₁₋₄alkyl, halo or cyano. In some of there embodiments, R^(11b) or R^(11c)is C₁₋₄ alkoxy, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄ cycloalkyl, NH—C₁₋₄alkyl, halo or cyano, and in further of these embodiments, R^(11b) isC₁₋₄ alkoxy, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄ cycloalkyl, NH—C₁₋₄alkyl, halo or cyano. Where one of R^(11a), R^(11b), R^(11c) and R^(11d)is C₁₋₄ alkoxy, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, C₃₋₄ cycloalkyl, NH—CH₁₋₄alkyl, halo or cyano, it may be that the group is methoxy, methyl, NHMe,F or cyano. In some embodiments, the group is methoxy.

R12

R^(12a), R^(12b), R^(12c) and R^(12d) are independently selected from H,F, CH₂OH and Me. In some of these embodiments, R^(12a), R^(12b), R^(12c)and R^(12d) are independently selected from H, Me and CH₂OH. In furtherof these embodiments, R^(12a), R^(12b), R^(12c) and R^(12d) areindependently selected from H and Me.

In some embodiments R^(12a), R^(12b), R^(12c) and R^(12d) are all H.

In some embodiments R^(12a), R^(12b), R^(12c) and R^(12d) are comprisedof three H and one Me or CH₂OH group. It may be preferred in theseembodiments that R^(12a) is Me and R^(12b), R^(12c) and R^(12d) are H.It may be preferred in these embodiments that R^(12c) is Me or CH₂OH andR^(12a), R^(12b) and R^(12d) are H.

In some embodiments R^(12a), R^(12b), R^(12c) and R^(12d) are comprisedof two H and two Me groups. It may be preferred in these embodimentsthat R^(12a) and R^(12c) are Me and R^(12b) and R^(12d) are H. It may bepreferred in these embodiments that R^(12a) and R^(12b) are Me andR^(12c) and R^(12d) are H. It may also be preferred in these embodimentsthat R^(12c) and R^(12d) are Me and R^(12a) and R^(12b) are H.

In some embodiments, R^(12c) and R^(12d) are independently selected fromH and F.

R^(12e)

In some embodiments, Rue is H. In other embodiments, Rue is Me.

R¹³

R^(13a) and R^(13b) are independently selected from H and Me. In someembodiments R^(13a) is H and R^(13b) is Me. In some embodiments R^(13a)and R^(13b) are both H. In some embodiments R^(13a) and R^(13b) are bothMe.

R¹⁴

In some embodiments R¹⁴ is H. In some embodiments R¹⁴ is Me.

R¹⁶

R^(16a) and R^(16b) are independently selected from H and Me. In someembodiments R^(16a) is H and R^(16b) is Me. In some embodiments R^(16a)and R^(16b) are both H. In some embodiments R^(16a) and R^(16b) are bothMe.

Stereoisomers

The carbon to which R¹⁴ is attached is a chiral centre.

In some embodiments, the compound is a mixture of stereoisomers at thiscentre. In some embodiments, the compound is a single stereoisomer. Insome of these embodiments, the compound is the (R)-stereoisomer. Inothers of these embodiments, the compound is the (S)-stereoisomer.

The compound may also include further chiral centres. The carbon in theTHIQ moiety is chiral. In some embodiments, the compound is a mixture ofstereoisomers at this centre. In other embodiments, the compound is asingle stereoisomer at this centre. In some of these embodiments, thecompound is the (R)-stereoisomer at this centre. In others of theseembodiments, the compound is the (S)-stereoisomer at this centre.

Thus the compound may be a single diastereomer or a mixture ofdiastereomers.

It may be preferred the compounds have the following stereochemistry:

Alternatively, the compounds may have one of the followingstereochemistries:

Furthermore, the compounds may have the following stereochemistry:

R¹¹-R¹⁵ n & p

In some embodiments, R^(11a), R^(11b), R^(11c), R^(11d), R^(12a),R^(12b), R^(12c), R^(12d), R^(12e), R¹⁴, R¹⁵, R^(16a) and R^(16b) areall H, n is 1 and p is 0, and thus the compound of formula I is offormula Id1:

In some embodiments, R^(11a), R^(11b), R^(11c), R^(11d), R^(12a),R^(12b), R^(12c), R^(12d), R^(12e), R^(13a), R^(13b), R¹⁴, R¹⁵, R^(16a)and R^(16b) are all H, n is 1 and p is 1, and thus the compound offormula I is of formula Id2:

In some embodiments, R^(11a), R^(11b), R^(11c), R^(11d), R^(12a),R^(12b), R^(12c), R^(12d), R^(12e), R¹⁴, R¹⁵, R^(16a) are all H, n is 1and p is 0, and R^(16b) is Me and thus the compound of formula I is offormula Id3:

R⁶

In some embodiments, R⁶ is H.

In some embodiments, R⁶ is OMe.

In some embodiments, R⁶ is OEt.

EXAMPLES

The following examples are provided solely to illustrate the presentinvention and are not intended to limit the scope of the invention, asdescribed herein.

Acronyms

For convenience, many chemical moieties are represented using well knownabbreviations, including but not limited to, methyl (Me), ethyl (Et),n-propyl (nPr), isopropyl (iPr), n-butyl (nBu), tert-butyl (tBu), phenyl(Ph), benzyl (Bn), methoxy (MeO), ethoxy (EtO), trimethylsilyl (TMS),and acetyl (Ac).

For convenience, many chemical compounds are represented using wellknown abbreviations, including but not limited to, methanol (MeOH),deuterated methanol (d₄-MeOD) ethanol (EtOH), isopropanol (i-PrOH),ether or diethyl ether (Et₂O), ethyl acetate (EtOAc), acetic acid(AcOH), acetonitrile (MeCN), dichloromethane (methylene chloride, DCM),trifluoroacetic acid (TFA), dimethylformamide (DMF), tetrahydrofuran(THF), dimethylsulfoxide (DMSO), deuterated chloroform (CDCl₃),diethylamine (DEA), deuterated dimethylsulfoxide (d₆-DMSO),N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl.HCl,EDCl), meta-chloroperoxybenzoic acid (mCPBA),1,1′-bis(diphenylphosphino)ferrocene (dppf), tert-butyloxycarbonyl (Boc,BOC), 2-(trimethylsilyl)ethoxymethyl (SEM), triethylamine (Et₃N or TEA),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), 4-dimethylaminopyridine (DMAP),N,N-diisopropylethylamine (DIPEA or DIEA),1,1′-bis(diphenylphosphino)ferrocene dichloropalladium (II)(PdCl₂(dppf)), trans-dichlorobis(triphenylphosphine)palladium(II)(PdCl₂(PPh₃)₂), tris(dibenzylideneacetone) dipalladium(0) (Pd₂(dba)₃),tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄), propylphosphonicanhydride (T3P), hexamethylphosphoramide (HMPA), 1,2-dichloroethane(DCE), benzyl (Bn) and 1-hydroxybenzotriazole (HOBt).

In addition, TLC refers to thin layer chromatography.

General Experimental Details

Unless otherwise stated the following generalisations apply. ¹H NMRspectra were recorded on a Bruker Ultrashield Plus (400 MHz) or a BrukerAVANCE (400 MHz). The multiplicity of a signal is designated by thefollowing abbreviations: s, singlet; d, doublet; t, triplet; q, quartet;dd, doublet of doublets; dt, doublet of triplets; tt, triplet oftriplets; br, broad; m, multiplet. All observed coupling constants, J,are reported in Hertz.

LCMS data was generated using either an Agilent 6100 Series Single QuadLCMS (LCMS-A), an Agilent 1260 Infinity Series UPLC/MS (LCMS-B), anAgilent 1200 Series G6110A Quadrupole LCMS or Waters 2695 alliance(LCMS-C). Chlorine isotopes are reported as ³⁵Cl, Bromine isotopes arereported as either ⁷⁹Br or ⁸¹Br or both ⁷⁹Br/⁸¹Br.

LCMS Method A (LCMS-A): Instrument: Agilent 6100 Series Single QuadLC/MS Agilent 1200 Series HPLC

Pump: 1200 Series G1311A Quaternary pump

Autosampler: 1200 Series G1329A Thermostatted Autosampler Detector: 1200Series G1314B Variable Wavelength Detector

LC conditions:Reverse Phase HPLC analysis

Column: Luna C₈ (2) 5 μm 50×4.6 mm 100 Å

Column temperature: 30° C.

Injection Volume: 5 μL Solvent A: Water 0.1% Formic Acid Solvent B: MeCN0.1% Formic Acid

Gradient: 5-100% solvent B over 10 min

Detection: 254 nm or 214 nm

MS conditions:

Ion Source: Quadrupole Ion Mode: Multimode-ES

Drying gas temp: 300° C.Vaporizer temperature: 200° C.Capillary voltage (V): 2000 (positive)Capillary voltage (V): 4000 (negative)

Scan Range: 100-1000

Step size: 0.1 secAcquisition time: 10 min

LCMS Method B (LCMS-B): Instrument: Agilent 1260 Infinity Series UPLC/MS

Pump: 1260 Infinity G1312B Binary pump

Autosampler: 1260 Infinity G1367E 1260 HiP ALS Detector: 1290 InfinityG4212A 1290 DAD

LC conditions:Reverse Phase HPLC analysis

Column: Poroshell 120 EC-C18 2.7 μm 50×3.0 mm

Column temperature: 35° C.

Injection Volume: 1 μL Solvent A: Water 0.1% Formic Acid Solvent B: MeCN0.1% Formic Acid

Gradient: 5-100% solvent B over 3.8 minDetection: monitored at 254 nm and 214 nmMS conditions:

Ion Source: Quadrupole Ion Mode: API-ES

Drying gas temp: 350° C.Capillary voltage (V): 3000 (positive)Capillary voltage (V): 3000 (negative)

Scan Range: 100-1000

Step size: 0.1 secAcquisition time: 5 min

LCMS Method C (LCMS-C): Instrument: Agilent 1200 Series G6110AQuadrupole

Pump: Binary pump

Detector: DAD

LC conditions:Reverse Phase HPLC analysis

Column: Xbridge-C18, 2.5 μm, 2.1×30 mm

Column temperature: 30° C.

Injection Volume: 1-10 μL

Solvent A: Water 0.07% Formic acid

Solvent B: Methanol

Gradient: 30-95% solvent B over 3.5 min (for medium polarity samples) or10-95% solvent B over 3.7 min (for large polarity samples)Detection: monitored at 254 nm and 214 nmMS conditions:

Ion Source: Quadrupole Ion Mode: ES+

Drying gas temp: 350° C.Drying gas flow: 10 L/minNebulizer pressure: 35 psiCapillary voltage (V): 3500 (positive)

Scan Range: 50-900 Or

Instrument: Waters 2695 alliance

Pump: Quaternary Pump Detector: 2996 Photodiode Array Detector

MS model: Micromass ZQLC conditions:

Column: Xbridge-C18, 3.5 μm, 2.1×50 mm

Column temperature: 30° C.Injection volume: 1-10 μLAcquisition of wavelength: 214 nm, 254 nmSolvent A: 0.07% HCOOH aqueous solution

Solvent B: MeOH

Run time: 8 minGradient: 20-95% solvent B over 5 min

Detection: 254 nm and 214 nm

MS condition:Ion source: ES+(or ES−) MS range: 50-900 m/z

Capillary: 3 kV Cone: 3 V Extractor: 3 V

Drying gas flow: 600 L/hr cone: 50 L/hrDesolvation temperature: 300° C.Source temperature: 100° C.

Sample Preparation:

The sample was dissolved in methanol, the concentration about 0.1-1.0mg/mL, then filtered through the syringes filter with 0.22 μm.

Preparative RP-HPLC:

Agilent 1260 Infinity HPLC systemUV detection at 210 nm and 254 nmGradient or isocratic elution through a Phenomenex Luna C8 (2) column100 Å Axia (250×21.2 mm; particle size 5 μm)Flow rate: 10 mL/minGradients are as specified in the individual examples.

Analytical thin-layer chromatography was performed on Merck silica gel60 F254 aluminium-backed plates which were visualised using fluorescencequenching under UV light or a basic KMnO₄ dip or Ninhydrin dip.

Preparative thin-layer chromatography (prep TLC) was performed usingTklst (China), grand grade: (HPTLC): 8±2 μm>80%; (TLC): 10-40 μm. Type:GF254. Compounds were visualised by UV (254 nm).

Flash chromatography was performed using a Biotage Isolera purificationsystem using either Grace or RediSep® silica cartridges.

Column chromatography was performed using Tklst (China), grand grade,100-200 meshes silica gel.

Microwave irradiation was achieved using a CEM Explorer SP MicrowaveReactor.

Where necessary, anhydrous solvents were purchased from Sigma-Aldrich ordried using conventional methods. Solutions of inorganic acids or baseswhere made up as aqueous solutions unless stated otherwise.

Additional Cartridges used are as follows:

Phase Separator:

Manufacturer: Biotage

Product: ISOLUTE Phase Separator (3 mL unless otherwise stated)

SCX and SCX-2 Cartridges:

Manufacturer: Biotage

Product: ISOLUTE SCX 1 g, (6 mL SPE Column unless otherwise stated)

Manufacturer: Biotage

Product: ISOLUTE SCX-2 1 g (6 mL Column)

Manufacturer: Silicycle

Product: SCX-2 500 mg or 5 g

Manufacturer: Agilent

Product: Bond Elute SCX 10 g

Sample Extraction Cartridge:

Manufacturer: Waters

Product: Oasis HLB 35 cc (6 g) LP extraction cartridge

Intermediate Preparations (i)(R)-tert-Butyl(3-amino-2-hydroxypropyl)(2,3-dihydro-1H-inden-2-yl)carbamate(I2)

(a)(R)-tert-Butyl(2,3-dihydro-1H-inden-2-yl)(3-(1,3-dioxoisoindolin-2-yl)-2-hydroxypropyl)carbamate(I1)

A mixture of 2,3-dihydro-1H-inden-2-amine (5.0 g, 24.61 mmol) and(R)-2-(oxiran-2-ylmethyl)isoindoline-1,3-dione (4.91 g, 36.91 mmol) inEtOH (100 mL) was heated at reflux overnight. After cooling to roomtemperature, triethylamine (9.94 g, 98.44 mmol) and (Boc)₂O (10.74 g,49.22 mmol) were added and the reaction mixture was stirred at roomtemperature overnight. The mixture was concentrated and purified bycolumn (EtOAc/petroleum ether=¼, v/v) to give the title compound (5.4 g,50%) as an oil.

(b)(R)-tert-Butyl(3-amino-2-hydroxypropyl)(2,3-dihydro-1H-inden-2-yl)carbamate(I2)

A mixture of(R)-tert-butyl(2,3-dihydro-1H-inden-2-yl)(3-(1,3-dioxoisoindolin-2-yl)-2-hydroxypropyl)carbamateI1 (4.4 g, 10.08 mmol) and NH₂NH₂.H₂O (80%, 1.89 g, 30.24 mmol) in EtOH(100 mL) was heated at reflux for 4 hours. After cooling to roomtemperature, the mixture was filtered and the filtercake was washed withEtOH (50 mL). The filtrate was concentrated and then dissolved inCH₂Cl₂, washed with saturated aqueous NaHCO₃ solution (80 mL×3), brine(20 mL×2), dried (Na₂SO₄), filtered and concentrated to give the titlecompound (3.1 g, crude) as a yellow oil, which was used for the nextstep without further purification. LCMS: RT 2.17 min; m/z 307.2 [M+H]⁺.

(ii)(R)-4((3-((tert-Butoxycarbonyl)(2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)carbamoyl)-3-ethoxybenzoicacid

(a) (R)-Methyl4-((3-((tert-butoxycarbonyl)(2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)carbamoyl)-3-ethoxybenzoate(I3)

To a solution of (R)-tert-butyl(3-amino-2-hydroxypropyl)(2,3-dihydro-1H-inden-2-yl) carbamate I2 (300mg, 0.98 mmol) and 2-ethoxy-4-(methoxycarbonyl)benzoic acid (220 mg,0.98 mmol) in DCM (10 mL) was added DIPEA (697 μL, 3.92 mmol), EDCl (387mg, 1.96 mmol) and HOBt (14 mg, 0.10 mmol). The reaction was stirred atroom temperature overnight, saturated aqueous NaHCO₃ solution (50 mL)was added, and the aqueous phase was extracted with DCM (3×50 mL), dried(Na₂SO₄), filtered and concentrated. The residue was purified by columnchromatography (petroleum ether:EtOAc=3:1) to give the desired product(370 mg, 74%) as a white solid. LCMS RT 3.23 min; m/z 513.3 [M+H]⁺

(b)(R)-4-((3-((tert-butoxycarbonyl)(2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)carbamoyl)-3-ethoxybenzoicacid (I4)

To a solution of (R)-methyl4-((3-((tert-butoxycarbonyl)(2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)carbamoyl)-3-ethoxybenzoateI3 (370 mg, 0.72 mmol) in a mixture of THF (10 mL), MeOH (3 mL) and H₂O(1 mL) was added LiOH.H₂O (91 mg, 2.16 mmol). The reaction was stirredat room temperature overnight. The mixture was concentrated underreduced pressure and the residue obtained suspended in water (15 mL).The mixture was acidified to pH 4 with 1 M aqueous HCl and the aqueousphase extracted with DCM (3×30 mL). The pooled organic extracts weredried (Na₂SO₄), filtered and concentrated to give the desired product(370 mg, 100%) as an off-white solid. LCMS RT 3.09 min; m/z 499.3 [M+H]+

(iii) tert-Butyl(S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I9)

(a)(S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (I5) (S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (5.00 g,28.2 mmol) was vigorously stirred in 1,4-dioxane (100 mL) and water (50mL). Sodium bicarbonate (4.74 mg, 56.4 mmol) and Boc anhydride (6.77 g,31.0 mmol) were added and the mixture was stirred vigorously at roomtemperature. After 17 hours the mixture was concentrated in vacuo andthe residue dissolved in water (200 mL). A 30% w/v aqueous solution ofsodium hydrogen sulfate monohydrate (30 mL) was added and the mixtureextracted with chloroform (3×200 mL). The pooled organic extracts werewashed with brine, dried over sodium sulfate and concentrated in vacuoto give the desired compound (7.50 g, 96% yield) as a thick syrup.LCMS-B: RT 3.64 min; m/z 178.1 [M-Boc+2H]⁺; m/z 276.1 [M−H]⁻

(b) tert-Butyl(S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I6)

(S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (I5) (7.50 g, 27.0 mmol) was dissolved in THF (150 mL) and CDI(8.77 g, 54.1 mmol) was added. The mixture was stirred for 30 minutes atroom temperature then cooled to 0° C. A solution of sodium borohydride(1.16 g, 30.5 mmol) in water (15 mL) was added dropwise. After 40minutes the mixture was quenched with acetone (25 mL) and concentratedin vacuo. The residue was partitioned between water (250 mL) and ethylacetate (200 mL). The separated aqueous phase was extracted with ethylacetate (2×250 mL), the combined organic extracts washed with 5% w/vaqueous NaHSO₄ (250 mL), brine (200 mL), dried over sodium sulfate andconcentrated in vacuo. The residue was loaded in diethyl ether (50 mL)onto a plug of basic alumina and silica (50 mL each). The plug waseluted with diethyl ether (250 mL) and the eluate evaporated to give thedesired compound (5.93 g, 83% yield) as a colourless syrup. ¹H NMR (400MHz, CDCl₃) δ 7.25-7.06 (m, 4H), 4.82-4.59 (m, 1H), 4.57-4.38 (m, 1H),4.37-4.19 (m, 1H), 3.57-3.40 (m, overlaps with trace solvent), 3.03 (dd,J=16.1, 5.7 Hz, 1H), 2.80 (d, J=16.1 Hz, 1H), 1.50 (s, 9H). LCMS-B: RT3.66 min; m/z 164.2 [M-Boc+2H]⁺, 286.2 [M+Na]⁺

(c) tert-Butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(I7)

tert-Butyl(S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I6)(1.50 g, 5.70 mmol), DCM (25 mL) and DMSO (5 mL) were cooled to 0° C.Triethylamine (2.38 mL, 17.1 mmol) was added, followed bypyridine-sulfur trioxide complex (2.72 g, 17.1 mmol). The mixture wasstirred at 0° C. for 10 minutes then allowed to come to roomtemperature. After 2 hours, saturated sodium bicarbonate (75 mL) andwater (75 mL) were added, and the mixture extracted with diethyl ether(3×150 mL). The pooled ether extracts were washed with 1:1 water:saturated aqueous NH₄Cl (200 mL), brine (200 mL), dried over sodiumsulfate, filtered and concentrated in vacuo to give the desired compoundas a colourless oil which was used without further purification.

(d) tert-Butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I8A) and tert-butyl(S)-3-((S)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I8B)

A solution of tert-butyl(S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I7) (5.70 mmol @100% conversion) in i-propanol (50 mL) was cooled to 0° C. Nitromethane(1.22 mL, 22.8 mmol) and potassium fluoride (331 mg, 5.70 mmol) wereadded and the mixture stirred for 18 hours, allowing the temperature tocome to room temperature as the ice bath thawed. The mixture was dilutedwith water (200 mL) and extracted with DCM (3×200 mL). The pooled DCMextracts were washed with brine, dried over sodium sulfate, filtered andconcentrated in vacuo. Chromatography (40 g silica cartridge, 0-20%ethyl acetate/hexanes) gave two partly overlapping peaks, which weresplit into early (8A major, colourless syrup, 697 mg, 37% yield) andlate (8B minor, colourless syrup, 170 mg, 9% yield) fractions. Overall:867 mg, 47% yield.

Data for major isomer tert-butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateI8A:

¹H NMR (400 MHz, d₄-MeOD) δ 7.25-7.14 (m, 4H), 4.85-4.49 (m, 5H), 4.44(dd, J=12.6, 9.3 Hz, 1H), 4.37-3.99 (m, overlaps with solvent), 3.19(dd, J=15.9, 3.2 Hz, 1H), 2.92 (dd, J=15.8, 5.6 Hz, 1H), 1.51 (s, 9H).LCMS-B: RT 3.71 min; m/z 223.2 [M-Boc+2H]⁺, 345.2 [M+Na]⁺; m/z 321.2[M−H]⁻

Data for minor isomer tert-butyl(S)-3-((S)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateI8B:

¹H NMR (400 MHz, d₄-MeOD) δ 7.25-7.11 (m, 4H), 4.75 (d, J=16.5 Hz, 1H),4.68-4.48 (m, 4H), 4.42-4.23 (m, overlaps with residual nitromethane),3.06 (dd, J=16.3, 6.1 Hz, 1H), 2.91 (d, J=16.1 Hz, 1H), 1.50 (s, 9H).LCMS-B: RT 3.70 min; m/z 223.2 [M-Boc+2H]⁺, 345.2 [M+Na]⁺; m/z 321.2[M−H]⁻

(e) tert-Butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I8A)

Copper catalyst used:

tert-Butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I7)(1.9 mmol @100% conversion), absolute ethanol (5 mL), nitromethane (1.02mL, 19.0 mmol) and the copper catalyst (91 mg, 10 mol %) (see abovefigure, prepared according to Tetrahedron:

Asymmetry (2008) 2310-2315) were stirred at room temperature. After 90hours the mixture was concentrated in vacuo, chromatography (40 g silicacartridge, 0-15% ethyl acetate/hexanes) gave the desired compound (352mg, 58% yield over two steps). ¹H NMR (400 MHz, d₄-MeOD) δ 7.25-7.13 (m,4H), 4.85-4.68 (m, 1H), 4.65-4.49 (m, 1H), 4.49-4.39 (m, 1H), 4.36-3.96(m, overlaps with trace solvent), 3.19 (dd, J=15.9, 3.2 Hz, 1H), 2.92(dd, J=15.9, 5.6 Hz, 1H), 1.51 (s, 9H). LCMS-B: RT 3.25 min; m/z 321.1[M−H]⁻

(f) tert-Butyl(S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I9)

tert-Butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I8A) (1.54 g, 4.78 mmol), absolute ethanol (75 mL) and 10% Pd/C (53%wetted with water, 1.5 g) were stirred under hydrogen (balloon). After 3hours the mixture was filtered through celite, the celite was washedwith absolute ethanol (100 mL) and the combined filtrates concentratedin vacuo to give the desired compound (1.34 g, 96% yield) as a palegrey-green syrup. LCMS-B: RT 3.27 min, m/z 293.2 [M+H]⁺

Alternate Synthesis Method (a)(S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (I5)

(S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (50.0 g, 282 mmol)was vigorously stirred in a mixture of 1,4-dioxane (1000 mL) and water(500 mL). Sodium bicarbonate (47.4 g, 564 mmol) and Boc anhydride (67.7g, 310 mmol) were added and the reaction was stirred vigorously at roomtemperature for 6 days. The mixture was concentrated in vacuo and theresidue dissolved in water (2000 mL). A 30% w/v aqueous solution ofsodium hydrogen sulfate monohydrate (300 mL) was added and the mixtureextracted with chloroform (3×1000 mL). The pooled organic extracts werewashed with brine, dried over sodium sulfate and concentrated in vacuoto give the desired compound (90.0 g, quantitative) as a thick syrup.LCMS-B: RT 3.64 min; m/z 178.1 [M-Boc+2H]⁺; m/z 276.1 [M−H]⁻

(b) tert-Butyl(S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I6)

(S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (I5) (54.0 g, 195 mmol) was dissolved in THF (1000 mL) and CDI(63.2 g, 390 mmol) was added. The mixture was stirred for 2 hours at 30°C. then cooled to 0° C. A solution of sodium borohydride (14.7 g, 390mmol) in water (120 mL) was added dropwise. After 3 hours the mixturewas quenched with acetone (300 mL) and concentrated in vacuo. Theresidue was partitioned between water (1000 mL) and ethyl acetate (1000mL). The separated aqueous phase was extracted with ethyl acetate (4×500mL) and the combined organic extracts washed with 5% w/v aqueous NaHSO₄(1000 mL), brine (500 mL), dried over sodium sulfate and concentrated invacuo. The residue was purified by chromatography (5-20% ethylacetate/petroleum ether) to give the desired compound (30.4 g, 59%yield) as a yellow syrup. ¹H NMR (400 MHz, CDCl₃) δ 7.25-7.06 (m, 4H),4.82-4.59 (m, 1H), 4.57-4.38 (m, 1H), 4.37-4.19 (m, 1H), 3.57-3.40 (m,overlaps with trace solvent), 3.03 (dd, J=16.1, 5.7 Hz, 1H), 2.80 (d,J=16.1 Hz, 1H), 1.50 (s, 9H). LCMS-B: RT 3.66 min; m/z 164.2[M-Boc+2H]⁺, 286.2 [M+Na]⁺

(c) tert-Butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate(I7)

tert-Butyl(S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I6) (16g, 0.06 mol), DCM (250 mL) and DMSO (75 mL) were cooled to 0° C.Triethylamine (25.1 mL, 0.18 mol) was added, followed by pyridine-sulfurtrioxide complex (28.6 g, 0.18 mol). The mixture was stirred at 0° C.for 30 minutes then allowed to come to room temperature and stirred atroom temperature overnight. Saturated sodium bicarbonate (200 mL) andwater (200 mL) were added, and the mixture extracted with diethyl ether(3×300 mL). The pooled ether extracts were washed with 1:1 water:saturated aqueous NH₄Cl (200 mL), dried over sodium sulfate andconcentrated in vacuo to give the desired compound (16.0 g) as an orangeoil which was used without further purification.

(e) tert-Butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I8A)

To a solution of tert-butyl(S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I7) (0.06 mol@100% conversion) in absolute ethanol (50 mL) was added a solution ofthe copper catalyst (6.8 g, 20 mol %) (see above figure, preparedaccording to Tetrahedron: Asymmetry (2008) 2310-2315) in absoluteethanol (10 mL). The mixture was cooled to 0° C. and nitromethane (36.0g, 0.6 mol) was added. The reaction was stirred at 0° C. for 3 days, themixture was concentrated in vacuo and purified by chromatography (5%ethyl acetate/petroleum ether) to give the desired compound (7.5 g, 39%yield over two steps). ¹H NMR (400 MHz, d₄-MeOD) δ 7.25-7.13 (m, 4H),4.85-4.68 (m, 1H), 4.65-4.49 (m, 1H), 4.49-4.39 (m, 1H), 4.36-3.96 (m,overlaps with trace solvent), 3.19 (dd, J=15.9, 3.2 Hz, 1H), 2.92 (dd,J=15.9, 5.6 Hz, 1H), 1.51 (s, 9H). LCMS-B: RT 3.25 min; m/z 321.1 [M−H]⁻

(f) tert-Butyl(S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I9)

To a solution of tert-butyl(S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I8A) (7.5 g, 23.3 mmol) in absolute ethanol (100 mL) was added 10% Pd/C(7.5 g) and the reaction was stirred under an atmosphere of hydrogen.After 3 hours, the mixture was filtered through Celite, the Celite waswashed with absolute ethanol (200 mL) and the combined filtratesconcentrated in vacuo to give the desired compound (5.3 g, 78% yield) asa pale grey solid. LCMS-B: RT 3.27 min, m/z 293.2 [M+H]⁺

(iv)4-(((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)carbamoyl)-3-ethoxybenzoicacid (I16)

(a) Ethyl-4-bromo-2-ethoxybenzoate (I10)

To a mixture of 4-bromo-2-hydroxybenzoic acid (20.0 g, 92.6 mmol) andK₂CO₃ (38.4 g, 278 mmol) in dimethylsulfoxide (50 mL) at 40° C. wasadded ethyl bromide (15.2 g, 139 mmol) dropwise over 30 minutes, thereaction mixture was stirred for 2 hours then further ethyl bromide(15.2 g, 139 mmol) was added over 30 minutes. The reaction was stirred afurther 8 hours then diluted with CH₂Cl₂ (150 mL) and filtered. Thefiltrate was washed with water (200 mL×10), brine (200 mL×3), dried(Na₂SO₄), filtered and concentrated to give the title compound as abrown solid (24.3 g, 96%): LCMS: RT 2.93 min; m/z 273.0 [M+H]⁺.

(b) 4-Bromo-2-ethoxybenzoic acid (I11)

To a solution of ethyl 4-bromo-2-ethoxybenzoate I10 (24.1 g, 88.6 mmol)in a mixture of THF (150 mL), methanol (15 mL) and water (15 mL) wasadded LiOH H₂O (18.6 g, 44.3 mmol). The resulting mixture was stirred atroom temperature for 24 hours. The solvent was removed, and the residuediluted with water (200 mL). The pH of the aqueous mixture was adjustedto 6 by addition of 2 M aqueous HCl. The aqueous mixture was extractedwith CH₂Cl₂ (150 mL×3) and the combined organic layers washed with brine(100 mL×3), dried (Na₂SO₄), filtered and concentrated to give the titlecompound as a yellow solid (19.8 g, 92%): LCMS: RT 2.47 min; m/z,244.9/246.9 [M+H]⁺.

(c) Benzyl-4-bromo-2-ethoxybenzoate (I12)

To a solution of 4-bromo-2-ethoxybenzoic acid I11 (16.4 g, 67.2 mmol) inacetonitrile (75 mL) was added benzyl bromide (13.8 g, 80.7 mmol) andK₂CO₃ (18.6 g, 134.4 mmol). The resulting mixture was stirred at 40° C.overnight. The reaction mixture was filtered and concentrated. Theresidue was diluted with CH₂Cl₂ (50 mL) and washed with water (100mL×2), brine (70 mL×3), dried (Na₂SO₄), filtered and concentrated. Thecrude product was purified by column chromatography (1% EtOAc inpetroleum ether) to give the title compound as a light-yellow oil (20.9g, 93%): LCMS: RT 3.31 min; m/z 334.9 [M+H]⁺.

(d) 1-Benzyl 4-methyl 2-ethoxyterephthalate (I13)

To a solution of benzyl 4-bromo-2-ethoxybenzoate I12 (20.0 g, 59.9 mmol)in methanol (100 mL) was added Pd(dppf)Cl₂ (2.2 g, 3 mmol) andtriethylamine (13.3 g, 131.7 mmol). The resulting mixture was heated atreflux overnight under an atmosphere of carbon monoxide. The reactionmixture was concentrated and the residue obtained was diluted with waterand was extracted with CH₂Cl₂ (50 mL×3). The combined organic layerswere washed with brine (60 mL×2), dried (Na₂SO₄), filtered andconcentrated. The residue was purified by column chromatography (2%EtOAc in petroleum ether) to give the title compound as a white solid(15.1 g, 81%): LCMS: RT 3.03 min; m/z 315.1 [M+H]⁺.

(e) 2-Ethoxy-4-(methoxycarbonyl)benzoic acid (I14)

To a solution of 1-benzyl 4-methyl 2-ethoxyterephthalate I13 (14.9 g,47.4 mmol) in THF (100 mL) was added 10% Pd/C (1.5 g). The resultingmixture was stirred at room temperature overnight under H2. The catalystwas removed by filtration through Celite and the filter cake washed withTHF (50 mL). The filtrate was concentrated and the residue was dilutedwith water. The pH of the aqueous solution was adjusted to 6 by additionof 2 M aqueous HCl and the resultant mixture extracted with CH₂Cl₂ (50mL×3). The combined organic layers were washed with brine (50 mL×2),dried (Na₂SO₄), filtered and concentrated to give the title compound asa white solid (10.2 g, 96%): LCMS: RT 2.13 min; m/z 225.0 [M+H]⁺.

(f)(S)-tert-Butyl-3-((R)-2-(2-ethoxy-4-(methoxycarbonyl)benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(I15)

To a solution of 2-ethoxy-4-(methoxycarbonyl)benzoic acid I14 (1.7 g,7.5 mmol) in CH₂Cl₂ (50 mL) were added (S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateI9 (2.0 g, 6.8 mmol), HOBt (100 mg, 0.7 mmol), DIPEA (3.9 g, 20.4 mmol)and EDCl (2.6 g, 13.6 mmol). The resulting mixture was stirred at roomtemperature overnight. The mixture was diluted with water (80 mL) andextracted with CH₂Cl₂ (80 mL×3). The combined organic layers were washedwith water (150 mL), brine (150 mL), dried (Na₂SO₄), filtered andconcentrated. The residue was purified by column chromatography (33%EtOAc in petroleum ether) to give the title compound as a yellow solid(2.2 g, 65%):

LCMS: RT 3.12 min; m/z 499.2 [M+H]⁺.

(g)4-(((R)-2-((S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyhcarbamoyl)-3-ethoxybenzoicacid (I16)

To a solution of(S)-tert-butyl-3-((R)-2-(2-ethoxy-4-(methoxycarbonyhbenzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate15 (2.1 g, 4.2 mmol) in methanol (4 mL) was added a solution of NaOH(340 mg, 8.4 mmol) in water (6 mL). The resulting mixture was stirred atroom temperature overnight. The solvent was removed, and the residuediluted with water (30 mL). The pH of the aqueous mixture was adjustedto 5 by addition of 2 M aqueous HCl solution. The aqueous layer was thenextracted with CH₂Cl₂ (50 mL×4) and the combined organic layers washedwith brine, dried (Na₂SO₄) and concentrated to give the title compoundas a yellow solid (1.9 g, 95%): LCMS: RT 2.95 min; m/z 485.2 [M+H]⁺.

(v) 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoic acidI20

(a) Ethyl-2-ethoxy-4-methylbenzoate I17

To a mixture of 2-hydroxy-4-methylbenzoic acid (8.2 g, 53.9 mmol) andK₂CO₃ (22.4 g, 161.7 mmol) in dimethylsulfoxide (70 mL) at 40° C. wasadded ethyl iodide (12.6 g, 80.8 mmol) dropwise over a period of 30 min.The reaction was stirred for 2 hours then further ethyl iodide (12.6 g,80.8 mmol) was added over 30 min. The resulting mixture was stirred afurther 8 hours at 40° C., diluted with CH₂Cl₂ (150 mL) and filtered.The filtrate was washed with water (200 mL×10) and brine (200 mL×2),dried (Na₂SO₄), filtered and concentrated to give the desired compoundas a yellow liquid (10.1 g, 90%): LCMS: RT 2.70 min; m/z 209.1 [M+H]⁺.

(b) 3-Ethoxy-4-(ethoxycarbonyl)-benzoic acid I18

To a solution of ethyl 2-ethoxy-4-methylbenzoate I17 (10.0 g, 48.1 mmol)in a mixture of pyridine (25 mL) and water (75 mL) was added KMnO₄ (22.8g, 144.2 mmol). The resulting mixture was heated at 50° C. for 48 hours,then cooled and allowed to stir at room temperature for 24 hours. Themixture was filtered and the filter cake washed with hot water. Thecombined aqueous filtrates were washed with EtOAc (75 mL×3) andacidified with 2M aqueous HCl solution. The mixture was extracted withCH₂Cl₂ (150 mL×3). The combined organic layers were washed with brine,dried (Na₂SO₄), filtered and concentrated to give the desired compoundas a white solid (5.0 g, 44%): LCMS: RT 0.25 min; m/z 239.0 [M+H]⁺.

(c) Ethyl4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoate I19

To a solution of 3-ethoxy-4-(ethoxycarbonyl)benzoic acid I18 (2.5 g,10.4 mmol) in CH₂Cl₂ (20 mL) was added 3-oxa-8-azabicyclo[3.2.1]octanehydrochloride (1.42 g, 9.5 mmol), HOBt (135.1 mg, 1.0 mmol), DIPEA (2.5g, 19.0 mmol) and EDCl (2.2 g, 11.4 mmol). The resulting mixture wasstirred at room temperature overnight. The mixture was partitioned withsaturated aqueous NaHCO₃, and extracted with CH₂Cl₂ (20 mL×2). Thecombined organic layers were washed with brine, dried (Na₂SO₄), filteredand concentrated. The residue was purified by column chromatography (1%methanol in dichloromethane) to give the desired compound as a yellowoil (2.5 g, 80%): LCMS: RT 2.40 min; m/z 334.1 [M+H]⁺.

(d) 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoic acidI20

To a solution ofethyl-4-(3-oxa-8-azabicyclo-[3.2.1]-octane-8-carbonyl)-2-ethoxybenzoateI19 (2.4 g, 7.2 mmol) in a mixture of THF (20 mL), methanol (2 mL) andwater (2 mL) was added LiOH H₂O (1.5 g, 36 mmol). The resulting mixturewas stirred at room temperature for 24 h. The solvent was removed, andthe residue obtained diluted with water (20 mL). The pH of the aqueousmixture was adjusted to 6 by addition of a 2 M aqueous HCl solution. Themixture was extracted with CH₂Cl₂ (20 mL×3) and the combined organiclayers washed with brine (10 mL×2), dried (Na₂SO₄), filtered andconcentrated to give the desired compound as a yellow oil (1.7 g, 79%):¹H NMR (400 MHz, d₄-MeOD) δ 7.83 (d, J=7.8 Hz, 1H), 7.19 (d, J=1.0 Hz,1H), 7.10 (dd, J=7.8, 1.3 Hz, 1H), 4.65 (br s, 1H), 4.20 (q, J=7.0 Hz,2H), 3.97 (br s, 1H), 3.82 (d, J=10.8 Hz, 1H), 3.72 (d, J=11.0 Hz, 2H),3.59 (d, J=10.9 Hz, 1H), 2.13-1.94 (m, 4H), 1.45 (t, J=7.0 Hz, 3H).LCMS:RT 1.20 min; m/z 306.1 [M+H]⁺.

EXAMPLES Example 1:2-Ethoxy-N-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-4-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamidehydrochloride (1)

(a) 2-(2,2-Diethoxyethoxy)-1,1-diethoxyethane (A1)

To a suspension of NaH (60% dispersion in mineral oil, 3.8 g, 95.0 mmol)in xylene (100 mL) was added 2,2-diethoxyethanol (11.6 g, 86.4 mmol)under N₂. The mixture heated at 110° C. for 2 hours, then cooled to roomtemperature and 2-bromo-1,1-diethoxyethane (25.6 g, 130.0 mmol) added.The reaction was heated at 110° C. overnight, then cooled to roomtemperature and the organic layer washed with water, dried (Na₂SO₄),filtered and concentrated. The residue obtained was purified bydistillation to give the title compound (7 g, 33%) as colorless oil.

(b) 9-Benzyl-3-oxa-9-azabicyclo[3.3.1]nonan-7-one (A2)

2-(2,2-Diethoxyethoxy)-1,1-diethoxyethane A1 (5 g, 20.0 mmol) in amixture of acetic acid (1.2 mL) and water (5 mL) was heated at refluxfor 1 hour then cooled to room temperature. Benzylamine hydrochloride(2.1 g, 20.0 mmoL) and 3-oxopentanedioic acid (2.5 g, 16.6 mmoL), NaOAc(0.69 g, 83.8 mmoL) and water (10 mL) were added and the reaction heatedat 50° C. for 5 hours. The reaction was cooled to room temperature andaqueous NaOH solution (42 mL, 50% w/v) was added into the mixture. Theaqueous phase was extracted with EtOAc (3×30 mL) and the combinedorganic fractions concentrated and purified by column chromatography(100% petroleum ether to 10% EtOAc in petroleum ether) to give the titlecompound as a yellow solid. LCMS: RT 0.50 min, m/z 232.1 [M+H]⁺

(c) tert-Butyl 7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (A3)

A mixture of 9-benzyl-3-oxa-9-azabicyclo[3.3.1]nonan-7-one A2 (425 mg,2.2 mmol), (Boc)₂O (1.3 g, 6.5 mmol) and 10% Pd/C (50 mg) in MeOH (10mL) was stirred vigorously under an atmosphere of H2 gas overnight. Thecatalyst was removed by filtration through celite and the filtrate wasconcentrated. The residue obtained was purified by column chromatography(100% petroleum ether to 10% EtOAc in petroleum ether) to give the titlecompound as a yellow solid (250 mg, 57%). LCMS: RT 0.53 min, m/z 264.1[M+Na]⁺.

(d) 3-Oxa-9-azabicyclo[3.3.1]nonan-7-one hydrochloride (A4)

A solution of tert-butyl7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate A3 (160 mg, 0.66mmol) in HCl in Et₂O (3 M solution, 3 mL) was stirred at roomtemperature for 2-3 hours. The mixture was concentrated and the crudeproduct obtained washed with ether to give the desired product as ayellow solid (120 mg, 100%). LCMS: RT 0.24 min, m/z 142.1 [M+H]⁺.

(e)(S)-tert-Butyl-3-((R)-2-(2-ethoxy-4-(7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(A5)

To a solution of -oxa-9-azabicyclo[3.3.1]nonan-7-one hydrochloride A4(80 mg, 0.37 mmol) and4-(((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)carbamoyl)-3-ethoxybenzoic acid I16 (151 mg, 0.31 mmol) in DCM (10 mL)was added EDCl (107 mg, 0.56 mmol), HOBt (76 mg, 0.56 mmol) and DIPEA(145 mg, 1.12 mmol). The reaction was stirred at room temperature for 3hours, then concentrated. The residue obtained was purified by columnchromatography (100% DCM to 10% MeOH in DCM) to give the title compoundas a yellow solid (150 mg, 79%). LCMS: RT 2.74 min, m/z 608.3 [M+H]⁺.

(f) (3S)-tert-Butyl3-((1R)-2-(2-ethoxy-4-(7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(A6)

A mixture of (S)-tert-butyl3-((R)-2-(2-ethoxy-4-(7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateA5 (110 mg, 0.18 mmol) in THF (5 mL) was added NaBH₄(34 mg, 0.90 mmol)and the reaction stirred at room temperature for 3 hours. The mixturewas concentrated and the residue obtained purified by columnchromatography (100% DCM to 10% MeOH in DCM) to give the title compoundas a white solid (70 mg, 63%). LCMS: RT 2.78 min, m/z 632.3 [M+Na]⁺. Therelative stereochemistry of the bicyclic alcohol was not determined.

(g)2-Ethoxy-N-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-4-(7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamidehydrochloride (1)

A solution of (3S)-tert-butyl3-((1R)-2-(2-ethoxy-4-(−7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateA6 (50 mg, 0.082 mmol) in a solution of HCl in Et₂O (3M, 5 mL) wasstirred at room temperature for 2-3 h. The mixture was concentrated andthe crude product washed with diethyl ether to give the desired productas a white solid (30 mg, 90%). ¹H NMR (400 MHz, CDCl₃) δ 7.97 (d, J=8.0Hz, 1H), 7.31-7.22 (m, 4H), 7.19 (d, J=0.8 Hz, 1H), 7.11 (dd, J=7.6 Hz,1.2 Hz, 1H), 4.68 (br s, 1H), 4.43 (q, J=7.2 Hz, 2H), 4.27-4.22 (m, 3H),4.0-4.97 (m, 2H), 3.89-3.85 (m, 3H), 3.76-3.70 (m, 2H), 3.66-3.59 (m,2H), 3.27 (br s, 1H), 3.22-3.17 (m, 1H), 2.39-2.33 (m, 1H), 2.23-2.18(m, 1H), 1.96 (m, 1H), 1.82-1.78 (m, 1H), 1.43 (t, J=7.2 Hz, 3H); LCMS:RT 3.25 min, m/z 510.3 [M+H]⁺.

Example 2:N-((S)-3-((2,3-Dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)-2-ethoxy-4-(−7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamide(2)

(a) tert-Butyl(2,3-dihydro-1H-inden-2-yl)((R)-3-(2-ethoxy-4-7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-2-hydroxypropyl)carbamate(A7)

To a solution of(R)-4-((3-((tert-butoxycarbonyl)(2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)carbamoyl)-3-ethoxybenzoicacid I4 (200 mg, 0.40 mmol) and 3-oxa-9-azabicyclo[3.3.1]nonan-7-onehydrochloride A4 (76 mg, 0.44 mmol) in DCM (5 mL) was added DIPEA (0.356mL, 2.00 mmol), EDCl (153 mg, 0.81 mmol) and HOBt (5 mg, 0.07 mmol). Theresultant mixture was stirred at room temperature overnight. Saturatedaqueous NaHCO₃ (30 mL) was added, and the mixture extracted with DCM(3×30 mL). The combined organic layers were dried (Na₂SO₄), filtered andconcentrated. The residue obtained was purified by preparative TLC(DCM:MeOH=10:1) to give the desired product (60 mg, 24%) as a greysolid. LCMS RT 2.85 min; m/z 622.3 [M+H]⁺

(b) tert-Butyl(2,3-dihydro-1H-inden-2-yl)((R)-3-(2-ethoxy-4-(−7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-2-hydroxypropyl)carbamate(A8)

To a solution of tert-butyl(2,3-dihydro-1H-inden-2-yl)((R)-3-(2-ethoxy-4-(7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-2-hydroxypropyl)carbamateA7 (60 mg, 0.10 mmol) in MeOH (5 mL) was added NaBH₄(11 mg, 0.30 mmol)and the reaction stirred at room temperature for 3 hours. The solventwas removed and the residue purified by preparative TLC (DCM:MeOH=10:1)to give the title compound (50 mg, 81%) as a white solid. LCMS: RT 1.31min; m/z 524.3 [M-Boc+2H]⁺.

(c)N-((S)-3-((2,3-Dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)-2-ethoxy-4-(7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamide(2)

tert-Butyl(2,3-dihydro-1H-inden-2-yl)((R)-3-(2-ethoxy-4-(−7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-2-hydroxypropyl)carbamateA8 (50 mg, 0.08 mmol) was stirred in a solution of 2.0 M solution of HClin Et₂O at room temperature for 2-3 hours. Saturated aqueous NaHCO₃ (30mL) was added, and the mixture was extracted with DCM (3×30 mL). Thecombined organic layers were dried (Na₂SO₄), filtered and concentrated.The reaction was repeated on another 60 mg of starting material and thecombined crude products were purified by preparative TLC (DCM:MeOH=10:1)provide the desired product (30 mg, 30%) as a white solid. ¹H NMR (400MHz, MeOD) δ 8.01 (d, J=8.0 Hz, 1H), 7.24-7.11 (m, 6H), 4.66 (m, 2H),4.29 (q, J=6.8 Hz, 2H), 4.06-3.76 (m, 7H), 3.63-3.59 (m, 1H), 3.54-3.49(m, 1H), 3.37-3.33 (m, 2H), 3.09-2.90 (m, 4H), 2.40-2.34 (m, 1H),2.25-2.18 (m, 1H), 2.04-1.95 (m, 1H), 1.83-1.79 (m, 1H), 1.53 (t, J=6.8Hz, 3H); LCMS: RT 1.26 min; m/z 524.3 [M+H]⁺.

Example 3:4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-N-methylbenzamidehydrochloride (3)

(a) (S)-tert-Butyl3-((R)-2-(benzylamino)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(A9)

To a solution of (S)-tert-butyl3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateI9 (200 mg, 0.68 mmol) in MeOH (2 mL) was added benzaldehyde (80 mg,0.75 mmol). The mixture was stirred at room temperature for 2 hours thenNaBH₄ (85 mg, 1.36 mmol) was added in portions. The mixture was stirredat room temperature for 2 hours then the solvent was removed and theresidue dissolved in water (20 mL). The solution was adjusted to pH 10by addition of 1.0 M aqueous NaOH solution and the aqueous layerextracted with DCM (3×20 mL). The combined organic extracts were washedwith brine (30 mL), dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by column chromatography (2% MeOH in DCM) to givethe title compound as a yellow oil (120 mg, 46%). LCMS: RT 2.33 min; m/z383.2 [M+H]⁺.

(b) (S)-tert-Butyl3-((R)-2-(benzyl(methyl)amino)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(A10)

To a solution of (S)-tert-butyl3-((R)-2-(benzylamino)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateA9 (120 mg, 0.31 mmol) in CH₃OH (2 mL) was added 37% aqueousformaldehyde (150 μL, 1.55 mmol), NaBH₃CN (40 mg, 0.62 mmol) and AcOH (1drop) and the reaction stirred at room temperature overnight. Thesolvent was removed and the residue dissolved in water (10 mL). The pHwas adjusted to 10 by addition of 0.5 M aqueous NaOH and the aqueousextracted with EtOAc (3×20 mL). The combined organic fractions weredried (Na₂SO₄) and concentrated. The residue was purified by preparativeTLC (5% MeOH in DCM) to give the title compound as yellow oil (90 mg,73%). LCMS: RT 2.23 min; m/z 397.2 [M+H]⁺.

(c) (S)-tert-Butyl3-((R)-1-hydroxy-2-(methylamino)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(A11)

A mixture of (S)-tert-butyl3-((R)-2-(benzyl(methyl)amino)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateA10 (80 mg, 0.20 mmol) and Pd(OH)₂ (14 mg, 0.1 mmol) in EtOAc (2 mL) wasstirred at 45° C. under a hydrogen atmosphere overnight. The catalystwas removed by filtration through Celite and the filtrate wasconcentrated. The residue obtained was purified by preparative TLC (7%MeOH/DCM) to give the title compound as a yellow oil (40 mg, 65%). LCMS:RT 2.19 min; m/z 307.2 [M+H]⁺

(d) (3S)-tert-Butyl3-((1R)-2-(4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N-methylbenzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate(A12)

To a solution of4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoic acid I20(40 mg, 0.13 mmol) and(S)-tert-butyl3-((R)-1-hydroxy-2-(methylamino)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateA11 (40 mg, 0.13 mmol) in DCM was added DIPEA (75 mg, 0.59 mmol), HOBt(2 mg, 0.013 mmol), and EDCl.HCl (50 mg, 0.26 mmol). The resultingmixture was stirred at room temperature for two days. The mixture wasdiluted with DCM (20 mL) and the organic layer washed with saturatedaqueous NaHCO₃ (20 mL), 0.5 M aqueous HCl (20 mL), water (20 mL) andbrine (20 mL), dried (Na₂SO₄), filtered and concentrated. The residueobtained was purified by preparative TLC (5% MeOH in DCM) to give thetitle compound as a yellow solid (35 mg, 45%). LCMS: RT 2.80 min; m/z594.3 [M+H]

(e)4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-N-methylbenzamidehydrochloride (3)

To a solution of (3S)-tert-butyl3-((1R)-2-(4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N-methylbenzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylateA12 (30 mg, 0.05 mmol) in Et₂O (1 mL) was added HCl in Et₂O (1.8 M, 1mL). The mixture was stirred at room temperature overnight. The solventwas removed and the solid washed with Et₂O (3×3 mL) to give the titlecompound as a yellow solid (23 mg, 85%). ¹H NMR (400 MHz, MeOD) δ7.34-7.32 (m, 5H), 7.14-7.06 (m, 2H), 4.62 (br s, 1H), 4.42-4.29 (m,3H), 4.09-3.59 (m, 11H), 3.22-3.19 (m, 1H), 3.04 (br s, 3H), 1.99 (br s,4H), 1.22 (br s, 3H), LCMS: RT 3.39 min; m/z 494.3 [M+H]⁺.

Assays PRMT5 Biochemical Assay

Compounds of the invention may be tested for in vitro activity in thefollowing assay: A histone H4 derived peptide is used as substrate(amino acid sequence:Ser-Gly-Arg-Gly-Lys-Gly-Gly-Lys-Gly-Leu-Gly-Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-NH₂).Full-length PRMT5 enzyme (NCBI Reference sequence NP_006100.2) wasco-expressed with His₆-MEP50 in insect cells and purified via Nickelimmobilized metal affinity and gel filtration chromatography (“theenzyme”).

The 6 μL reactions are run in Greiner brand black 384-well low volumeassay plates. All reactions contained assay buffer (phosphate bufferedsaline, 0.01% (v/v) Tween-20, 0.01% (w/v) albumin from chicken eggwhite, 1 mM Dithiothreitol, 1 μM peptide substrate, 1 μM S-Adenosylmethionine, and 15 ng/reaction enzyme, with the enzyme being omittedfrom negative control reactions. Compounds were added in a volume of 100nL from dilution series prepared in DMSO, positive and negative controlreactions receiving the same volume DMSO without compound. The plateswere sealed with adhesive seals and incubated for 4 hours at 37 degreeCelsius. Reaction progress was measured using the Transcreener™ EPIGENmethyltransferase assay (BellBrook Labs, Madison, Wis.) as recommendedby the manufacturer. To each reaction 2 μL detection mix were added,containing coupling enzymes, fluorescence polarisation tracer, and AMPantibody. Plates were incubated for 90 minutes before being read on aPerkinElmer EnVision™ plate reader in fluorescence polarisation mode.IC₅₀ values were obtained from the raw readings by calculating percentinhibition (% I) for each reaction relative to controls on the sameplate (% I=(I−CN)/(CP−CN) where CN/CP are the averages of thenegative/positive reactions, respectively), then fitting the % I datavs. compound concentration [I] to % I=(A+((B−A)/(1+((C/[I]{circumflexover ( )}D)))) where A is the lower asymptote, B is the upper asymptote,C is the IC₅₀ value, and D is the slope.

Compound Number IC₅₀ (μM) 1 0.019 2 0.019 3 0.036

PRMT5 Biomarker Assay

Compounds of the invention may be tested for potency to inhibitsymmetrical dimethylation of arginine in the following assay:

The cell line TE11 was seeded at a density of 6,000 cells per well in 96well optical quality tissue culture plates in DME medium and 10% foetalbovine serum, and allowed to adhere for 5 hours under standard cultureconditions (37 degree Celsius, 5% CO₂). Compound dilutions prepared inDMSO were added to the medium, with negative control wells reserved fortreatment with DMSO only and maximum inhibition controls receiving apotent PRMT5 inhibitor compound at 1 μM concentration. After incubationfor 72 hours, the cells were fixed with 3.7% formaldehyde in PBS for 30minutes at room temperature, washed with phosphate buffer saline andblocked with Odyssey blocking buffer (LI-COR, Lincoln, Nebr.). Rabbitanti-Di-Methyl Histone H4 Arginine 3 specific antibody (Epigentek) inOdyssey blocking buffer was added and incubated for 14 hours at 4 degreeCelsius. After washing, anti-rabbit secondary antibody labelled withAlexa647 dye (LifeTechnologies) and Hoechst 33342 (1 μg/mL,SigmaAldrich) were added for 1 hour incubation. Plates were washed andread on a PerkinElmer Envision 2103 in fluorescence intensity scanningmode (24 scans across the well area). The plates were imaged on aPerkinElmer Phenix high content imaging platform. Using a Columbus imageanalysis pipeline, individual nuclei were located by Hoechst 33342 stainand the methylation level was calculated from the Alexa647-relatedintensity in the same area. IC₅₀ values were obtained from the meanAlexa647-related intensity per cell by calculating percent inhibition (%I) for each well relative to controls on the same plate (%I=(I−CN)/(CP−CN) where CN/CP are the averages of the negative/maximuminhibition controls, respectively), then fitting the % I data vs.compound concentration [I] to % I=(A+((B−A)/(1+((C/[I]){circumflex over( )}D)))) where A is the lower asymptote, B is the upper asymptote, C isthe IC₅₀ value, and D is the slope.

Compound Number IC₅₀ (μM) 1 0.0039 2 0.0006 3 0.0117

1-72. (canceled)
 73. A compound of formula:

74-81. (canceled)
 82. A method for the treatment of cancer orhemoglobinopathies, comprising administering to a patient in need oftreatment, a therapeutically effective PRMT5 activity inhibiting amountof a composition comprising a compound of formula Ia, Ib, Ic or Id:

wherein: n is 1 or 2; R^(N) is H or Me; R¹ (if present) is one or morehalo or methyl groups; R^(2a) and R^(2b) are independently selected fromthe group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH₂OH; R^(2c)and R^(2d) (if present) are independently selected from the groupconsisting of: (i) F; (ii) H; (iii) Me; and (iv) CH₂OH; R^(3a) andR^(3b) are independently selected from H and Me; R^(4a) is selected fromOH, —NH₂, —C(═O)NH₂, and —CH₂OH; R^(4b) is either H or Me; R⁵ is eitherH or Me; m is 1 or 2; q is 0 or 1; R^(11a), R^(11b), R^(11c) and R^(11d)are independently selected from H, halo, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl,C₃₋₄ cycloalkyl, C₁₋₄ alkyloxy, NH—C₁₋₄ alkyl and cyano; R^(12a) andR^(12b) are independently selected from the group consisting of: (i) F;(ii) H; (iii) Me; and (iv) CH₂OH; R^(12c) and R^(12d) are independentlyselected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv)CH₂OH; R^(12e) is H or Me; R^(13a) and R^(13b) are independentlyselected from H and Me; R¹⁴ is either H or Me; R^(16a) and R^(16b) areindependently selected from H and Me; R⁶ is selected from H, OMe, andOEt; or pharmaceutically acceptable salts thereof; and a pharmaceuticalcarrier.
 83. The method according to claim 82, wherein n is
 1. 84. Themethod according to claim 82, wherein R^(N) is H.
 85. The methodaccording to claim 82, wherein there are no R¹ substituents.
 86. Themethod according to claim 82, wherein R^(2a), R^(2b), R^(2c) and R^(d)(if present) are all H.
 87. The method according to claim 82, whereinR^(3a) and R^(3b) are both H.
 88. The method according to claim 82,wherein R^(4a) is OH.
 89. The method according to claim 82, whereinR^(4b) is H.
 90. The method according to claim 82, wherein R⁵ is H. 91.The method according to claim 82 which is of formula Ia1, Ib1 or Ic1:


92. The method according to claim 82, wherein the compound is the(S)-enantiomer at the carbon atom to which R^(4b) and R^(4a) areattached.
 93. The method according to claim 82, wherein m is
 1. 94. Themethod according to claim 93, wherein q is
 0. 95. The method accordingto claim 92, wherein R^(11a), R^(11b), R^(11c) and R^(11d) are all H.96. The method according to claim 92, wherein R^(12a), R^(12b), R^(12c)and R^(12d) are all H.
 97. The method according to claim 92, whereinR^(13a) and R^(13b) are both H.
 98. The method according to claim 92,wherein R¹⁴ is H.
 99. The method according to claim 92, wherein R^(16a)and R^(16b) are both H.
 100. The method according to claim 92, whereinR⁵ is H.
 101. The method according to claim 82 which is of formula Id1:


102. The method according to claim 92, which has the followingstereochemistry:


103. The method according to claim 82, wherein R⁶ is OEt.
 104. Themethod according to claim 82, which is selected from:2-Ethoxy-N-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-4-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamidehydrochloride (1); andN-((S)-3-((2,3-Dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)-2-ethoxy-4-(−7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamide(2).
 105. The method of claim 82, wherein the cancer is any one or moreof the following: leukemia, acute lymphocytic leukemia (ALL), acutemyeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronicmyeloid leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's disease,prostate cancer, lung cancer, melanoma, breast cancer, colon and rectalcancer, colon cancer, squamous cell carcinoma and gastric cancer.